Tolerogenic synthetic nanocarriers for inducing regulatory B cells

ABSTRACT

Disclosed are synthetic nanocarrier methods, and related compositions, comprising B cell and/or MHC Class II-restricted epitopes and immunosuppressants in order to generate tolerogenic immune responses, such as the generation of antigen-specific regulatory B cells.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119 of U.S.provisional application 61/480,946, filed Apr. 29, 2011, 61/513,514,filed Jul. 29, 2011, 61/531,147, filed Sep. 6, 2011, 61/531,153, filedSep. 6, 2011, 61/531,164, filed Sep. 6, 2011, 61/531,168, filed Sep. 6,2011, 61/531,175, filed Sep. 6, 2011, 61/531,180, filed Sep. 6, 2011,61/531,194, filed Sep. 6, 2011, 61/531,204, filed Sep. 6, 2011,61/531,209, filed Sep. 6, 2011, 61/531,215, filed Sep. 6, 2011, theentire contents of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to methods of administering synthetic nanocarriercompositions with immunosuppressants and B cell and/or MHC ClassII-restricted epitopes of an antigen wherein the compositions cangenerate regulatory B cells, and related compositions. The methods allowfor efficient uptake by APCs to shift the immune response in favor ofregulatory B cell development specific to antigens.

BACKGROUND OF THE INVENTION

Conventional strategies for generating immunosuppression associated withan undesired immune response are based on broad-acting immunosuppressivedrugs. Additionally, in order to maintain immunosuppression,immunosuppressant drug therapy is generally a life-long proposition.Unfortunately, the use of broad-acting immunosuppressants are associatedwith a risk of severe side effects, such as tumors, infections,nephrotoxicity and metabolic disorders. Accordingly, newimmunosuppressant therapies would be beneficial.

SUMMARY OF THE INVENTION

In one aspect, a method comprising administering to a subject acomposition that comprises: (i) a first population of syntheticnanocarriers coupled to immunosuppressants, and (ii) a second populationof synthetic nanocarriers coupled to comprise B cell and/or MHC ClassII-restricted epitopes of an antigen is provided. In another aspect, amethod comprising generating antigen-specific regulatory B cells in asubject by administering a composition that comprises (i) a firstpopulation of synthetic nanocarriers coupled to immunosuppressants, and(ii) a second population of synthetic nanocarriers coupled to B celland/or MHC Class II-restricted epitopes of an antigen is provided. Inanother aspect, a method comprising: administering to a subject acomposition according to a protocol that was previously shown togenerate antigen-specific regulatory B cells in one or more testsubjects; wherein the composition comprises: (i) a first population ofsynthetic nanocarriers coupled to immunosuppressants, and (ii) a secondpopulation of synthetic nanocarriers coupled to B cell and/or MHC ClassII-restricted epitopes of an antigen is provided. In one embodiment, thecomposition is in an amount effective to generate antigen-specificregulatory B cells in the subject. In another embodiment, thecomposition is administered in an amount effective to generateantigen-specific regulatory B cells in the subject.

In one embodiment, the first population and the second population arethe same population. In another embodiment, the first population andsecond population are different populations.

In another embodiment, the method further comprises providing oridentifying the subject.

In another embodiment, the antigen is a therapeutic protein, anautoantigen or an allergen, or is associated with an inflammatorydisease, an autoimmune disease, organ or tissue rejection or graftversus host disease.

In another embodiment, the method further comprises assessing thegeneration of antigen-specific regulatory B cells in the subject priorto and/or after the administration of the composition.

In another embodiment, the subject has or is at risk of having aninflammatory disease, an autoimmune disease, an allergy, organ or tissuerejection or graft versus host disease. In another embodiment, thesubject has undergone or will undergo transplantation. In anotherembodiment, the subject has or is at risk of having an undesired immuneresponse against a therapeutic protein that is being administered orwill be administered to the subject.

In another embodiment, one or more maintenance doses of the compositioncomprising the first population and second population of syntheticnanocarriers is administered to the subject.

In another embodiment, the administering is by intravenous,intraperitoneal, transmucosal, oral, subcutaneous, pulmonary,intranasal, intradermal or intramuscular administration. In anotherembodiment, the administering is by inhalation or intravenous,subcutaneous or transmucosal administration.

In another embodiment, the method further comprises administering atransplantable graft or therapeutic protein. In another embodiment, theadministering of the transplantable graft or therapeutic protein, whenthe therapeutic protein is provided as one or more cells, is byparenteral, intraarterial, intranasal or intravenous administration orby injection to lymph nodes or anterior chamber of the eye or by localadministration to an organ or tissue of interest.

In another embodiment, the method further comprises collecting thegenerated antigen-specific regulatory B cells.

In another embodiment, the immunosuppressants comprise a statin, an mTORinhibitor, a TGF-β signaling agent, a corticosteroid, an inhibitor ofmitochondrial function, a P38 inhibitor, an NF-κβ inhibitor, anadenosine receptor agonist, a prostaglandin E2 agonist, aphosphodiesterasse 4 inhibitor, an HDAC inhibitor or a proteasomeinhibitor. In another embodiment, the mTOR inhibitor is rapamycin or arapamycin analog.

In another embodiment, the load of the immunosuppressants and/orantigens on average across the first and/or second population ofsynthetic nanocarriers is between 0.0001% and 50% (weight/weight). Inanother embodiment, the load of the immunosuppressants and/or antigenson average across the first and/or second population of syntheticnanocarriers is between 0.1% and 10% (weight/weight).

In another embodiment, the synthetic nanocarriers of the firstpopulation and/or second population comprise lipid nanoparticles,polymeric nanoparticles, metallic nanoparticles, surfactant-basedemulsions, dendrimers, buckyballs, nanowires, virus-like particles orpeptide or protein particles. In another embodiment, the syntheticnanocarriers of the first population and/or second population compriselipid nanoparticles. In another embodiment, the synthetic nanocarriersof the first population and/or second population comprise liposomes. Inanother embodiment, the synthetic nanocarriers of the first populationand/or second population comprise metallic nanoparticles. In anotherembodiment, the metallic nanoparticles comprise gold nanoparticles. Inanother embodiment, the synthetic nanocarriers of the first populationand/or second population comprise polymeric nanoparticles. In anotherembodiment, the polymeric nanoparticle comprises polymer that is anon-methoxy-terminated, pluronic polymer. In another embodiment, thepolymeric nanoparticles comprise a polyester, a polyester coupled to apolyether, polyamino acid, polycarbonate, polyacetal, polyketal,polysaccharide, polyethyloxazoline or polyethyleneimine. In anotherembodiment, the polyester comprises a poly(lactic acid), poly(glycolicacid), poly(lactic-co-glycolic acid) or polycaprolactone. In anotherembodiment, the polymeric nanoparticles comprise a polyester and apolyester coupled to a polyether. In another embodiment, the polyethercomprises polyethylene glycol or polypropylene glycol.

In another embodiment, the mean of a particle size distribution obtainedusing dynamic light scattering of the synthetic nanocarriers of thefirst and/or second population is a diameter greater than 100 nm. Inanother embodiment, the diameter is greater than 150 nm. In anotherembodiment, the diameter is greater than 200 nm. In another embodiment,the diameter is greater than 250 nm. In another embodiment, the diameteris greater than 300 nm. In another embodiment, the aspect ratio of thesynthetic nanocarriers of the first population and/or second populationis greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7 or 1:10.

In another embodiment, the method further comprises making a dosage formcomprising the collected antigen-specific regulatory B cells. In anotherembodiment, the method further comprises making the collectedantigen-specific regulatory B cells available or dosage form availableto a subject for administration.

In another embodiment, the second population of synthetic nanocarriersare also coupled to antigens that comprise MHC I-restricted epitopes.

In another aspect, a composition comprising the isolatedantigen-specific regulatory B cells produced according to any of themethods provided herein is provided.

In another embodiment, the composition further comprises apharmaceutically acceptable excipient.

In another aspect, a dosage form comprising the any of the compositionsprovided herein is provided.

In another aspect, a composition comprising isolated antigen-specificregulatory B cells for use in therapy or prophylaxis, said isolatedantigen-specific regulatory B cells being obtainable by a processcomprising the steps of: (a) generating antigen-specific regulatory Bcells in a subject by administering a composition that comprises: (i) afirst population of synthetic nanocarriers coupled toimmunosuppressants, and (ii) a second population of syntheticnanocarriers coupled to B cell and/or MHC Class II-restricted epitopesof an antigen; and (b) collecting the generated antigen-specificregulatory B cells is provided. In one embodiment, the composition isany of the compositions provided herein or used in any of the methodsprovided.

In another aspect, a composition comprising: (i) a first population ofsynthetic nanocarriers coupled to immunosuppressants, and (ii) a secondpopulation of synthetic nanocarriers coupled to B cell and/or MHC ClassII-restricted epitopes of an antigen, for use in therapy or prophylaxisis provided. In one embodiment, the composition is any of thecompositions provided herein or used in any of the methods provided.

In another aspect, any of the compositions provided may be for use intherapy or prophylaxis.

In another aspect, any of the compositions provided may be for use inpromoting a tolerogenic immune response or generating antigen-specificregulatory B cells in a subject.

In another aspect, a use of any of the compositions or dosage formsprovided for the manufacture of a medicament for use in promoting atolerogenic immune response or generating antigen-specific regulatory Bcells in a subject is provided.

In another embodiment, the composition further comprises atransplantable graft or therapeutic protein.

In another aspect, a dosage form comprising any of the compositionsprovided is provided.

In another aspect, a method comprising: (i) producing a first populationof synthetic nanocarriers coupled to immunosuppressants, and (ii)producing a second population of synthetic nanocarriers coupled to Bcell and/or MHC Class II-restricted epitopes of an antigen is provided.In one embodiment, the composition is in an amount effective to generateantigen-specific regulatory B cells in a subject. In another embodiment,the first population and second population are the same population. Inanother embodiment, the first population and second population aredifferent populations.

In another embodiment, the method further comprises making a dosage formcomprising the first population and second population of syntheticnanocarriers.

In another embodiment, the method further comprises making a compositioncomprising the first population and second population of syntheticnanocarriers or the dosage form available to a subject foradministration.

In another embodiment, the method further comprises assessing thegeneration of antigen-specific regulatory B cells with a compositioncomprising the first population and second population of syntheticnanocarriers.

In another embodiment, the first population and second population ofsynthetic nanocarriers that are produced are as defined in any of thecompositions and methods provided herein.

In another aspect, a process for producing a composition or dosage formcomprising the steps of: (i) coupling a first population of syntheticnanocarriers to immunosuppressants, and (ii) coupling a secondpopulation of synthetic nanocarriers to B cell and/or MHC ClassII-restricted epitopes of an antigen is provided. In one embodiment, thecomposition or dosage form is in an amount effective to generateregulatory B cells in a subject. In one embodiment, the steps are asdefined in any of the methods provided herein.

In another aspect, a composition or dosage form obtainable by any of themethods or processes provided herein is provided.

In another embodiment, the methods further comprise including atransplantable graft or therapeutic protein with the collectedantigen-specific regulatory B cells or dosage form.

In another embodiment, the first and/or second population of syntheticnanocarriers of any of the compositions or methods provided are alsocoupled to MHC Class I-restricted epitopes. In an embodiment of any ofthe compositions and methods provided herein, the antigens comprisesubstantially no MHC Class I-restricted epitopes.

In an embodiment of any of the compositions and methods provided herein,antigens that are proteins that comprise the aforementioned epitopes canbe coupled to the synthetic nanocarriers. In another embodiment,polypeptides or peptides that comprise the aforementioned epitopes butadditional amino acids that flank one or both ends of the epitope(s) canbe coupled to the synthetic nanocarriers. In another embodiment, theepitopes themselves are coupled to the synthetic nanocarriers.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 provides a representative example of a flow cytometric analysisof Treg cells.

FIG. 2 demonstrates that synthetic nanocarriers of the inventionresulted in higher proportions of antigen-specific Bregs.

FIG. 3 shows a decrease in IgE production with synthetic nanocarriers ofthe invention.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified materials or process parameters as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments of the inventiononly, and is not intended to be limiting of the use of alternativeterminology to describe the present invention.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entiretyfor all purposes.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a polymer”includes a mixture of two or more such molecules or a mixture ofdiffering molecular weights of a single polymer species, reference to “asynthetic nanocarrier” includes a mixture of two or more such syntheticnanocarriers or a plurality of such synthetic nanocarriers, reference to“a DNA molecule” includes a mixture of two or more such DNA molecules ora plurality of such DNA molecules, reference to “an immunosuppressant”includes a mixture of two or more such materials or a plurality of animmunosuppressant molecules, and the like.

As used herein, the term “comprise” or variations thereof such as“comprises” or “comprising” are to be read to indicate the inclusion ofany recited integer (e.g. a feature, element, characteristic, property,method/process step or limitation) or group of integers (e.g. features,element, characteristics, properties, method/process steps orlimitations) but not the exclusion of any other integer or group ofintegers. Thus, as used herein, the term “comprising” is inclusive anddoes not exclude additional, unrecited integers or method/process steps.

In embodiments of any of the compositions and methods provided herein,“comprising” may be replaced with “consisting essentially of” or“consisting of”. The phrase “consisting essentially of” is used hereinto require the specified integer(s) or steps as well as those which donot materially affect the character or function of the claimedinvention. As used herein, the term “consisting” is used to indicate thepresence of the recited integer (e.g. a feature, element,characteristic, property, method/process step or limitation) or group ofintegers (e.g. features, element, characteristics, properties,method/process steps or limitations) alone.

A. Introduction

Delivering immunosuppressants and antigens via synthetic nanocarriercompositions that comprise B cell and/or MHC Class II-restrictedepitopes more directly at the sites of action in cells of interest, inparticular APCs, can stimulate regulatory B cells and result inbeneficial tolerogenic immune responses specific to the antigens. Asshown in the examples, synthetic nanocarrier compositions were found tobe effective in generating CD24+ B cells that produce IL-10 and TGF-βand also recognize antigen, which speaks to the antigen-specific natureof the response. Additionally, the same synthetic nanocarriercompositions were found to decrease IgE production, which suggests adownstream result of the regulatory B cell production. The compositionsprovided, without being bound to any particular theory, can providetolerogenic immune effects directly as a result of the recognition ofepitopes, which may cause the generation of the regulatory cells, theswitching of B cells to a regulatory phenotype, etc. The tolerogenicimmune responses may also be a result of the production of cytokinesand/or the production of other regulatory immune cells stimulated bysuch cytokines. The generation of regulatory B cells can also lead todownstream tolerogenic effects. For example, regulatory B cells canreduce the proliferative capacity of effector T cells, such as CD4+ Tcells, and/or enhance FoxP3 and CTLA-4 expression in regulatory T cells.Regulatory B cells can also produce the regulatory cytokine, IL-10. Inembodiments, the antigen-specific itDCs provided herein may have theseeffects, for example, on a subject.

The generation of such regulatory B cells evidences the ability of thecompositions of the invention to generate antigen-specific tolerogenicimmune responses that can have utility in the treatment or prophylaxisof a variety of diseases, disorders or conditions. Therefore, thisinvention is useful, for example, to promote tolerogenic immuneresponses in subjects who have or are at risk of having an allergy,autoimmune disease, an inflammatory disease, organ or tissue rejectionor graft versus host disease. This invention is also useful forpromoting tolerogenic immune responses in subjects who have undergone orwill undergo transplantation. This invention is also useful forpromoting tolerogenic immune responses in subjects that have received,are receiving or will receive a therapeutic protein against whichundesired immune responses are generated or are expected to begenerated. The present invention, in some embodiments, prevents orsuppresses undesired immune responses that may neutralize the beneficialeffect of certain therapeutic treatments.

The inventors have unexpectedly and surprisingly discovered that theproblems and limitations noted above can be overcome by practicing theinvention disclosed herein. In particular, the inventors haveunexpectedly discovered that it is possible to provide syntheticnanocarrier compositions, and related methods, that induce a tolerogenicimmune response through the increase in the number and/or activity ofregulatory B cells. The method described herein include administering toa subject a composition that comprises (i) a first population ofsynthetic nanocarriers coupled to immunosuppressants, and (ii) a secondpopulation of synthetic nanocarriers coupled to B cell and/or MHC ClassII-restricted epitopes of an antigen. Preferably, the composition is inan amount effective to effect antigen-specific regulatory B cellproliferation and/or activity in the subject. In another aspect, amethod comprising generating antigen-specific regulatory B cells in asubject by administering a composition that comprises (i) a firstpopulation of synthetic nanocarriers coupled to immunosuppressants, and(ii) a second population of synthetic nanocarriers coupled to B celland/or MHC Class II-restricted epitopes of an antigen is provided. Inanother aspect, a method comprising administering to a subject acomposition according to a protocol that was previously shown togenerate antigen-specific regulatory B cells in one or more testsubjects wherein the composition comprises (i) a first population ofsynthetic nanocarriers coupled to immunosuppressants, and (ii) a secondpopulation of synthetic nanocarriers coupled to B cell and/or MHC ClassII-restricted epitopes of an antigen is provided.

Transplantable grafts, therapeutic proteins, etc. may also beadministered to the subjects as provided herein. Such compositions maybe administered to a subject prior to, concomitantly with or after theadministration of the first and second populations of syntheticnanocarriers. Such additional agents may or may not be coupled to thefirst or second population of synthetic nanocarriers or anotherpopulation of synthetic nanocarriers. In embodiments, the compositionsprovided may also be administered as one or more maintenance doses to asubject. In such embodiments, the compositions provided are administeredsuch that the generation of a desired immune response is maintained oran undesired immune response is reduced for a certain length of time.Examples of such lengths of time are provided elsewhere herein.

In another aspect, a composition comprising isolated antigen-specificregulatory B cells produced according to any of the methods provided isprovided. In yet another aspect, the compositions comprising the firstpopulation and second population of synthetic nanocarriers are alsoprovided. In embodiments, the compositions further comprise atransplantable graft or a therapeutic protein that may or may not becoupled to the first or second population of synthetic nanocarriers oranother population of synthetic nanocarriers.

In another aspect, dosage forms of any of the compositions herein areprovided. Such dosage forms may be administered to a subject, forexample, one in need of antigen-specific regulatory B cell generation.

In yet another aspect, a method of (i) producing a first population ofsynthetic nanocarriers coupled to immunosuppressants, and (ii) producinga second population of synthetic nanocarriers coupled to B cell and/orMHC Class II-restricted epitopes of an antigen is provided. In oneembodiment, the method further comprises producing a dosage formcomprising the first and second populations of synthetic nanocarriers.In another embodiment, the method further comprises ensuring the secondpopulation of synthetic nanocarriers comprises B cell and/or MHC ClassII-restricted epitopes of an antigen. In one embodiment, the presence ofa B cell epitope and/or MHC Class II-restricted can be confirmed by theability of such epitope to stimulate immune responses such as antibodyproduction or B cell proliferation and/or activity for the B cellepitopes and CD4+ T cell immune responses for the MHC ClassII-restricted epitopes. Methods for determining these activity are wellknown to those of ordinary skill in the art or are provided elsewhereherein. In still another embodiment, the method further comprisesassessing the generation of regulatory B cells with a composition ordosage form comprising the first population and second population ofsynthetic nanocarriers. In one embodiment, this assessment is performedin vitro. In another embodiment, this assessment is performed in vivo,such as with a sample obtained from a subject. In yet anotherembodiment, the method further comprises making a composition comprisingthe first population and second population of synthetic nanocarriers orthe dosage form available to a subject for administration.

In another aspect, the compositions or dosage forms produced by any ofthe methods provided herein are also provided.

The invention will now be described in more detail below.

B. Definitions

“Administering” or “administration” means providing a material to asubject in a manner that is pharmacologically useful.

“Allergens” are any substances that can cause an undesired (e.g., a Type1 hypersensitive) immune response (i.e., an allergic response orreaction) in a subject. Allergens include, but are not limited to, plantallergens (e.g., pollen, ragweed allergen), insect allergens, insectsting allergens (e.g., bee sting allergens), animal allergens (e.g., petallergens, such as animal dander or cat Fel d 1 antigen), latexallergens, mold allergens, fungal allergens, cosmetic allergens, drugallergens, food allergens, dust, insect venom, viruses, bacteria, etc.Food allergens include, but are not limited to milk allergens, eggallergens, nut allergens (e.g., peanut or tree nut allergens, etc.(e.g., walnuts, cashews, etc.)), fish allergens, shellfish allergens,soy allergens, legume allergens, seed allergens and wheat allergens.Insect sting allergens include allergens that are or are associated withbee stings, wasp stings, hornet stings, yellow jacket stings, etc.Insect allergens also include house dust mite allergens (e.g., Der P1antigen) and cockroach allergens. Drug allergens include allergens thatare or are associated with antibiotics, NSAIDs, anaesthetics, etc.Pollen allergens include grass allergens, tree allergens, weedallergens, flower allergens, etc. Subjects that develop or are at riskof developing an undesired immune response to any of the allergensprovided herein may be treated with any of the compositions and methodsprovided herein. Subjects that may be treated with any of thecompositions and methods provided also include those who have or are atrisk of having an allergy to any of the allergens provided.

An “allergy” also referred to herein as an “allergic condition,” is anycondition where there is an undesired (e.g., a Type 1 hypersensitive)immune response (i.e., allergic response or reaction) to a substance.Such substances are referred to herein as allergens. Allergies orallergic conditions include, but are not limited to, allergic asthma,hay fever, hives, eczema, plant allergies, bee sting allergies, petallergies, latex allergies, mold allergies, cosmetic allergies, foodallergies, allergic rhinitis or coryza, topic allergic reactions,anaphylaxis, atopic dermatitis, hypersensitivity reactions and otherallergic conditions. The allergic reaction may be the result of animmune reaction to any allergen. In some embodiments, the allergy is afood allergy. Food allergies include, but are not limited to, milkallergies, egg allergies, nut allergies, fish allergies, shellfishallergies, soy allergies or wheat allergies.

“Amount effective” in the context of a composition or dosage form foradministration to a subject refers to an amount of the composition ordosage form that produces one or more desired immune responses in thesubject, for example, the generation of regulatory B cells. Therefore,in some embodiments, an amount effective is any amount of a compositionprovided herein that produces such a desired immune response. Thisamount can be for in vitro or in vivo purposes. For in vivo purposes,the amount can be one that a clinician would believe may have a clinicalbenefit for a subject in need of antigen-specific tolerization. Suchsubjects include those that have or are at risk of having aninflammatory disease, an autoimmune disease, an allergy, organ or tissuerejection or graft versus host disease. Such subjects also include thosethat have undergone or will undergo transplantation. Such subjectsfurther include those that have experienced, are experiencing or areexpected to experience an undesired immune response against atherapeutic protein. Other subjects include those described elsewhereherein.

Amounts effective can involve only reducing the level of an undesiredimmune response, although in some embodiments, it involves preventing anundesired immune response altogether. Amounts effective can also involvedelaying the occurrence of an undesired immune response. Amountseffective can also involve generating a desired immune response. Anamount that is effective can also be an amount of a composition providedherein that produces a desired therapeutic endpoint or a desiredtherapeutic result. Amounts effective, preferably, result in atolerogenic immune response in a subject to an antigen. Preferably, theamounts effective result in the increased proliferation and/or activityof regulatory B cells. The achievement of any of the foregoing can bemonitored by routine methods.

In some embodiments of any of the compositions and methods provided, theamount effective is one in which the desired immune response persists inthe subject for at least 1 week, at least 2 weeks, at least 1 month, atleast 2 months, at least 3 months, at least 4 months, at least 5 months,at least 6 months, at least 9 months, at least 1 year, at least 2 years,at least 5 years, or longer. In other embodiments of any of thecompositions and methods provided, the amount effective is one whichproduces a measurable desired immune response, for example, a measurabledecrease in an immune response (e.g., to a specific antigen), for atleast 1 week, at least 2 weeks, at least 1 month, at least 2 months, atleast 3 months, at least 4 months, at least 5 months, at least 6 months,at least 9 months, at least 1 year, at least 2 years, at least 5 years,or longer.

Amounts effective will depend, of course, on the particular subjectbeing treated; the severity of a condition, disease or disorder; theindividual patient parameters including age, physical condition, sizeand weight; the duration of the treatment; the nature of concurrenttherapy (if any); the specific route of administration and like factorswithin the knowledge and expertise of the health practitioner. Thesefactors are well known to those of ordinary skill in the art and can beaddressed with no more than routine experimentation. It is generallypreferred that a maximum dose be used, that is, the highest safe doseaccording to sound medical judgment. It will be understood by those ofordinary skill in the art, however, that a patient may insist upon alower dose or tolerable dose for medical reasons, psychological reasonsor for virtually any other reason.

In general, doses of the immunosuppressants and/or antigens in thecompositions of the invention can range from about 10 μg/kg to about100,000 μg/kg. In some embodiments, the doses can range from about 0.1mg/kg to about 100 mg/kg. In still other embodiments, the doses canrange from about 0.1 mg/kg to about 25 mg/kg, about 25 mg/kg to about 50mg/kg, about 50 mg/kg to about 75 mg/kg or about 75 mg/kg to about 100mg/kg. Alternatively, the dose can be administered based on the numberof synthetic nanocarriers that provide the desired amount ofimmunosuppressants and/or antigens. For example, useful doses includegreater than 10⁶, 10⁷, 10⁸, 10⁹ or 10¹⁰ synthetic nanocarriers per dose.Other examples of useful doses include from about 1×10⁶ to about 1×10¹⁰,about 1×10⁷ to about 1×10⁹ or about 1×10⁸ to about 1×10⁹ syntheticnanocarriers per dose.

“Antigen” means a B cell antigen or T cell antigen. “Type(s) ofantigens” means molecules that share the same, or substantially thesame, antigenic characteristics. In some embodiments, antigens may beproteins, polypeptides, peptides, lipoproteins, glycolipids,polynucleotides, polysaccharides or are contained or expressed in cells.In some embodiments, such as when the antigens are not well defined orcharacterized, the antigens may be contained within a cell or tissuepreparation, cell debris, cell exosomes, conditioned media, etc. Anantigen can be combined with the synthetic nanocarriers in the same formas what a subject is exposed to that causes an undesired immune responsebut may also be a fragment or derivative thereof. When a fragment orderivative, however, a desired immune response to the form encounteredby such a subject is the preferable result with the compositions andmethods provided. Preferably, the antigens comprise epitopes such thatregulatory B cells are generated, recruited or activated with thecompositions provided herein. Whether or not this occurs can beestablished through measurement of cytokine production (e.g., IL-10)and/or regulatory B cell proliferation using conventional techniques.

“Antigen-specific” refers to any immune response that results from thepresence of the antigen, or portion thereof, or that generates moleculesthat specifically recognize or bind the antigen. For example, where theimmune response is antigen-specific antibody production, antibodies areproduced that specifically bind the antigen. As another example, wherethe immune response is antigen-specific regulatory B cell proliferationand/or activity, the proliferation and/or activity results fromrecognition of the antigen, or portion thereof, alone or in complex withMHC molecules, by B cells, etc.

“Antigens associated” with a disease, disorder or condition providedherein are antigens that can generate an undesired immune responseagainst, as a result of, or in conjunction with the disease, disorder orcondition; the cause of the disease, disorder or condition (or a symptomor effect thereof); and/or can generate an undesired immune responsethat is a symptom, result or effect of the disease, disorder orcondition. Preferably, in some embodiments, the use of an antigenassociated with a disease, disorder or condition, etc. in thecompositions and methods provided herein will lead to a tolerogenicimmune response against the antigen and/or the cells, by, on or in whichthe antigen is expressed. The antigens can be in the same form asexpressed in a subject with the disease, disorder or condition but mayalso be a fragment or derivative thereof. When a fragment or derivative,however, a desired immune response to the form expressed in such asubject is the preferable result with the compositions and methodsprovided.

In one embodiment, the antigen is an antigen associated with aninflammatory disease, autoimmune disease, organ or tissue rejection orgraft versus host disease. Such antigens include autoantigens, such asmyelin basic protein, collagen (e.g., collagen type 11), human cartilagegp 39, chromogranin A, gp130-RAPS, proteolipid protein, fibrillarin,nuclear proteins, nucleolar proteins (e.g., small nucleolar protein),thyroid stimulating factor receptor, histones, glycoprotein gp 70,ribosomal proteins, pyruvate dehydrogenase dehydrolipoamideacetyltransferase, hair follicle antigens, human tropomyosin isoform 5,mitochondrial proteins, pancreatic β-cell proteins, myelinoligodendrocyte glycoprotein, insulin, glutamic acid decarboxylase(GAD), gluten, and fragments or derivatives thereof. Other autoantigensare provided in Table 1 below.

Antigens also include those associated with organ or tissue rejection.Examples of such antigens include, but are not limited to, antigens fromallogeneic cells, e.g., antigens from an allogeneic cell extract andantigens from other cells, such as endothelial cell antigens.

Antigens also include those associated with an allergy. Such antigensinclude the allergens described elsewhere herein.

Antigens also include those associated with a transplantable graft. Suchantigens are associated with a transplantable graft, or an undesiredimmune response in a recipient of a transplantable graft that isgenerated as a result of the introduction of the transplantable graft inthe recipient, that can be presented for recognition by cells of theimmune system and that can generate an undesired immune response.Transplant antigens include those associated with organ or tissuerejection or graft versus host disease. Transplant antigens may beobtained or derived from cells of a biological material or frominformation related to a transplantable graft. Transplant antigensgenerally include proteins, polypeptides, peptides, lipoproteins,glycolipids, polynucleotides or are contained or expressed in cells.Information related to a transplantable graft is any information about atransplantable graft that can be used to obtain or derive transplantantigens. Such information includes information about antigens thatwould be expected to be present in or on cells of a transplantable graftsuch as, for example, sequence information, types or classes of antigensand/or their MHC Class I, MHC Class II or B cell presentationrestrictions. Such information may also include information about thetype of transplantable graft (e.g, autograft, allograft, xenograft), themolecular and cellular composition of the graft, the bodily locationfrom which the graft is derived or to which the graft to be transplanted(e.g., whole or partial organ, skin, bone, nerves, tendon, neurons,blood vessels, fat, cornea, etc.).

Antigens also include antigens associated with a therapeutic proteinthat can be presented for recognition by cells of the immune system andthat can generate an undesired immune response against the therapeuticprotein. Therapeutic protein antigens generally include proteins,polypeptides, peptides, lipoproteins, or are contained or expressed in,by or on cells.

Antigens, can be antigens that are fully defined or characterized.However, in some embodiments, an antigen is not fully defined orcharacterized. Antigens, therefore, also include those that arecontained within a cell or tissue preparation, cell debris, cell exosomeor conditioned media and can be delivered in such form in someembodiments.

“Assessing an immune response” refers to any measurement ordetermination of the level, presence or absence, reduction, increase in,etc. of an immune response in vitro or in vivo. Such measurements ordeterminations may be performed on one or more samples obtained from asubject. Such assessing can be performed with any of the methodsprovided herein or otherwise known in the art.

An “at risk” subject is one in which a health practitioner believes hasa chance of having a disease, disorder or condition as provided hereinor is one a health practitioner believes has a chance of experiencing anundesired immune response as provided herein.

An “autoimmune disease” is any disease where the immune system mounts anundesired immune response against self (e.g., one or more autoantigens).In some embodiments, an autoimmune disease comprises an aberrantdestruction of cells of the body as part of the self-targeted immuneresponse. In some embodiments, the destruction of self manifests in themalfunction of an organ, for example, the colon or pancreas. Examples ofautoimmune diseases are described elsewhere herein. Additionalautoimmune diseases will be known to those of skill in the art and theinvention is not limited in this respect.

“Average”, as used herein, refers to the arithmetic mean unlessotherwise noted.

“B cell antigen” means any antigen that triggers an immune response in aB cell (e.g., an antigen that is specifically recognized by a B cell ora receptor thereon). In some embodiments, an antigen that is a T cellantigen is also a B cell antigen. In other embodiments, the T cellantigen is not also a B cell antigen. B cell antigens include, but arenot limited to proteins, peptides, small molecules, and carbohydrates.In some embodiments, the B cell antigen comprises a non-protein antigen(i.e., not a protein or peptide antigen). In some embodiments, the Bcell antigen comprises a autoantigen. In other embodiments, the B cellantigen is obtained or derived from an allergen, autoantigen,therapeutic protein, or transplantable graft.

“Concomitantly” means administering two or more substances to a subjectin a manner that is correlated in time, preferably sufficientlycorrelated in time so as to provide a modulation in an immune response.In embodiments, concomitant administration may occur throughadministration of two or more substances in the same dosage form. Inother embodiments, concomitant administration may encompassadministration of two or more substances in different dosage forms, butwithin a specified period of time, preferably within 1 month, morepreferably within 1 week, still more preferably within 1 day, and evenmore preferably within 1 hour.

“Couple” or “Coupled” or “Couples” (and the like) means to chemicallyassociate one entity (for example a moiety) with another. In someembodiments, the coupling is covalent, meaning that the coupling occursin the context of the presence of a covalent bond between the twoentities. In non-covalent embodiments, the non-covalent coupling ismediated by non-covalent interactions including but not limited tocharge interactions, affinity interactions, metal coordination, physicaladsorption, host-guest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof. In embodiments,encapsulation is a form of coupling.

“Derived” means prepared from a material or information related to amaterial but is not “obtained” from the material. Such materials may besubstantially modified or processed forms of materials taken directlyfrom a biological material. Such materials also include materialsproduced from information related to a biological material.

“Dosage form” means a pharmacologically and/or immunologically activematerial in a medium, carrier, vehicle, or device suitable foradministration to a subject.

“Encapsulate” means to enclose at least a portion of a substance withina synthetic nanocarrier. In some embodiments, a substance is enclosedcompletely within a synthetic nanocarrier. In other embodiments, most orall of a substance that is encapsulated is not exposed to the localenvironment external to the synthetic nanocarrier. In other embodiments,no more than 50%, 40%, 30%, 20%, 10% or 5% (weight/weight) is exposed tothe local environment. Encapsulation is distinct from absorption, whichplaces most or all of a substance on a surface of a syntheticnanocarrier, and leaves the substance exposed to the local environmentexternal to the synthetic nanocarrier.

“Epitope”, also known as an antigenic determinant, is the part of anantigen that is recognized by the immune system, specifically by, forexample, antibodies, B cells, or T cells. As used herein, “MHC ClassI-restricted epitopes” are epitopes that are presented to immune cellsby MHC class I molecules found on nucleated cells. “MHC ClassII-restricted epitopes” are epitopes that are presented to immune cellsby MHC class II molecules found on antigen-presenting cells (APCs), forexample, on professional antigen-presenting immune cells, such as onmacrophages, B cells, and dendritic cells, or on non-hematopoieticcells, such as hepatocytes. “B cell epitopes” are molecular structuresthat are recognized by antibodies or B cells. In some embodiments, theepitope itself is an antigen.

A number of epitopes are known to those of skill in the art, andexemplary epitopes suitable according to some aspects of this inventioninclude, but are not limited to those listed in the Immune EpitopeDatabase (www.immuneepitope.org, Vita R, Zarebski L, Greenbaum J A,Emami H, Hoof I, Salimi N, Damle R, Sette A, Peters B. The immuneepitope database 2.0. Nucleic Acids Res. 2010 January; 38(Databaseissue):D854-62; the entire contents of which as well as all databaseentries of IEDB version 2.4, August 2011, and particularly all epitopesdisclosed therein, are incorporated herein by reference). Epitopes canalso be identified with publicly available algorithms, for example, thealgorithms described in Wang P, Sidney J, Kim Y, Sette A, Lund O,Nielsen M, Peters B. 2010. peptide binding predictions for HLA DR, DPand DQ molecules. BMC Bioinformatics 2010, 11:568; Wang P, Sidney J, DowC, Motile B, Sette A, Peters B. 2008. A systematic assessment of MHCclass II peptide binding predictions and evaluation of a consensusapproach. PLoS Comput Biol. 4(4):e1000048; Nielsen M, Lund O. 2009.NN-align. An artificial neural network-based alignment algorithm for MHCclass II peptide binding prediction. BMC Bioinformatics. 10:296; NielsenM, Lundegaard C, Lund O. 2007. Prediction of MHC class II bindingaffinity using SMM-align, a novel stabilization matrix alignment method.BMC Bioinformatics. 8:238; Bui H H, Sidney J, Peters B, Sathiamurthy M,Sinichi A, Purton K A, Mothé B R, Chisari F V, Watkins D I, Sette A.2005. Immunogenetics. 57:304-314; Sturniolo T, Bono E, Ding J,Raddrizzani L, Tuereci O, Sahin U, Braxenthaler M, Gallazzi F, Protti MP, Sinigaglia F, Hammer J. 1999. Generation of tissue-specific andpromiscuous HLA ligand databases using DNA microarrays and virtual HLAclass II matrices. Nat. Biotechnol. 17(6):555-561; Nielsen M, LundegaardC, Worning P, Lauemoller S L, Lamberth K, Buus S, Brunak S, Lund 0.2003.Reliable prediction of T-cell epitopes using neural networks with novelsequence representations. Protein Sci 12:1007-1017; Bui H H, Sidney J,Peters B, Sathiamurthy M, Sinichi A, Purton K A, Mothe B R, Chisari F V,Watkins D I, Sette A. 2005. Automated generation and evaluation ofspecific MHC binding predictive tools: ARB matrix applications.Immunogenetics 57:304-314; Peters B, Sette A. 2005. Generatingquantitative models describing the sequence specificity of biologicalprocesses with the stabilized matrix method. BMC Bioinformatics 6:132;Chou P Y, Fasman G D. 1978. Prediction of the secondary structure ofproteins from their amino acid sequence. Adv Enzymol Relat Areas MolBiol 47:45-148; Emini E A, Hughes J V, Perlow D S, Boger J. 1985.Induction of hepatitis A virus-neutralizing antibody by a virus-specificsynthetic peptide. J Virol 55:836-839; Karplus P A, Schulz G E. 1985.Prediction of chain flexibility in proteins. Naturwissenschaften72:212-213; Kolaskar A S, Tongaonkar P C. 1990. A semi-empirical methodfor prediction of antigenic determinants on protein antigens. FEBS Lett276:172-174; Parker J M, Guo D, Hodges R S. 1986. New hydrophilicityscale derived from high-performance liquid chromatography peptideretention data: correlation of predicted surface residues withantigenicity and X-ray-derived accessible sites. Biochemistry25:5425-5432; Larsen J E, Lund O, Nielsen M. 2006. Improved method forpredicting linear B-cell epitopes. Immunome Res 2:2; Ponomarenko J V,Bourne P E. 2007. Antibody-protein interactions: benchmark datasets andprediction tools evaluation. BMC Struct Biol 7:64; Haste Andersen P,Nielsen M, Lund 0.2006. Prediction of residues in discontinuous B-cellepitopes using protein 3D structures. Protein Sci 15:2558-2567;Ponomarenko J V, Bui H, Li W, Fusseder N, Bourne P E, Sette A, Peters B.2008. ElliPro: a new structure-based tool for the prediction of antibodyepitopes. BMC Bioinformatics 9:514; Nielsen M, Lundegaard C, Blicher T,Peters B, Sette A, Justesen S, Buus S, and Lund 0.2008. PLoS ComputBiol. 4(7)e1000107. Quantitative predictions of peptide binding to anyHLA-DR molecule of known sequence: NetMHCIIpan; the entire contents ofeach of which are incorporated herein by reference for disclosure ofmethods and algorithms for the identification of epitopes.

Other examples of epitopes include any of the MHC Class I-restricted,MHC Class II-restricted and B cell epitopes as provided as SEQ ID NOs:1-943. Without wishing to being bound by any particular theory, MHCClass I-restricted epitopes include those set forth in SEQ ID NOs:1-186, MHC Class II-restricted epitopes include those set forth in SEQID NOs: 187-537, and B cell epitopes include those set forth in SEQ IDNOs: 538-943. These epitopes include MHC Class I-restrictedautoantigens, MHC Class II-restricted epitopes of allergens and B cellepitopes of autoantigens and allergens.

“Generating” means causing an action, such as an immune response (e.g.,a tolerogenic immune response) to occur, either directly oneself orindirectly, such as, but not limited to, an unrelated third party thattakes an action through reliance on one's words or deeds.

“Identifying” is any action or set of actions that allows a clinician torecognize a subject as one who may benefit from the methods andcompositions provided herein. Preferably, the identified subject is onewho is in need of a tolerogenic immune response as provided herein. Theaction or set of actions may be either directly oneself or indirectly,such as, but not limited to, an unrelated third party that takes anaction through reliance on one's words or deeds.

“Immunosuppressant” means a compound that causes an APC to have animmunosuppressive (e.g., tolerogenic effect). An immunosuppressiveeffect generally refers to the production or expression of cytokines orother factors by the APC that reduces, inhibits or prevents an undesiredimmune response but can also include the production or expression ofcytokines or other factors by the APC that increases, stimulates orpromotes a desired immune response. When the APC results in animmunosuppressive effect on immune cells that recognize an antigenpresented by the APC, the immunosuppressive effect is said to bespecific to the presented antigen. Such effect is also referred toherein as a tolerogenic effect. Without being bound by any particulartheory, it is thought that the immunosuppressive or tolerogenic effectis a result of the immunosuppressant being delivered to the APC,preferably in the presence of an antigen (e.g., an administered antigenor one that is already present in vivo). Accordingly, theimmunosuppressant includes compounds that provide a tolerogenic immuneresponse to an antigen that may or may not be provided in the samecomposition or a different composition. In one embodiment, theimmunosuppressant is one that causes an APC to promote a regulatoryphenotype in one or more immune effector cells. For example, theregulatory phenotype may be characterized by the production, induction,stimulation or recruitment of Treg cells, etc. This may be the result ofthe conversion of B cells to a regulatory phenotype. This may also bethe result of the stimulation, recruitment, etc. of regulatory B cells.This may also be the result of induction of FoxP3 in other immune cells,such as macrophages and iNKT cells. In one embodiment, theimmunosuppressant is one that affects the response of the APC after itprocesses an antigen. In another embodiment, the immunosuppressant isnot one that interferes with the processing of the antigen. In a furtherembodiment, the immunosuppressant is not an apoptotic-signalingmolecule. In another embodiment, the immunosuppressant is not aphospholipid.

Immunosuppressants include, but are not limited to, statins; mTORinhibitors, such as rapamycin or a rapamycin analog; TGF-β signalingagents; TGF-β receptor agonists; histone deacetylase inhibitors, such asTrichostatin A; corticosteroids; inhibitors of mitochondrial function,such as rotenone; P38 inhibitors; NF-κβ inhibitors, such as 6Bio,Dexamethasone, TCPA-1, IKK VII; adenosine receptor agonists;prostaglandin E2 agonists (PGE2), such as Misoprostol; phosphodiesteraseinhibitors, such as phosphodiesterase 4 inhibitor (PDE4), such asRolipram; proteasome inhibitors; kinase inhibitors; G-protein coupledreceptor agonists; G-protein coupled receptor antagonists;glucocorticoids; retinoids; cytokine inhibitors; cytokine receptorinhibitors; cytokine receptor activators; peroxisomeproliferator-activated receptor antagonists; peroxisomeproliferator-activated receptor agonists; histone deacetylaseinhibitors; calcineurin inhibitors; phosphatase inhibitors; PI3 KBinhibitors, such as TGX-221; autophagy inhibitors, such as3-Methyladenine; aryl hydrocarbon receptor inhibitors; proteasomeinhibitor I (PSI); and oxidized ATPs, such as P2X receptor blockers.Immunosuppressants also include IDO, vitamin D3, cyclosporins, such ascyclosporine A, aryl hydrocarbon receptor inhibitors, resveratrol,azathiopurine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG),FK506, sanglifehrin A, salmeterol, mycophenolate mofetil (MMF), aspirinand other COX inhibitors, niflumic acid, estriol and triptolide. Inembodiments, the immunosuppressant may comprise any of the agentsprovided herein.

The immunosuppressant can be a compound that directly provides theimmunosuppressive (e.g., tolerogenic) effect on APCs or it can be acompound that provides the immunosuppressive (e.g., tolerogenic) effectindirectly (i.e., after being processed in some way afteradministration). Immunosuppressants, therefore, include prodrug forms ofany of the compounds provided herein.

Immunosuppressants also include nucleic acids that encode the peptides,polypeptides or proteins provided herein that result in animmunosuppressive (e.g., tolerogenic) immune response. In embodiments,therefore, the immunosuppressant is a nucleic acid that encodes apeptide, polypeptide or protein that results in an immunosuppressive(e.g., tolerogenic) immune response, and it is the nucleic acid that iscoupled to the synthetic nanocarrier.

The nucleic acid may be DNA or RNA, such as mRNA. In embodiments, theinventive compositions comprise a complement, such as a full-lengthcomplement, or a degenerate (due to degeneracy of the genetic code) ofany of the nucleic acids provided herein. In embodiments, the nucleicacid is an expression vector that can be transcribed when transfectedinto a cell line. In embodiments, the expression vector may comprise aplasmid, retrovirus, or an adenovirus amongst others. Nucleic acids canbe isolated or synthesized using standard molecular biology approaches,for example by using a polymerase chain reaction to produce a nucleicacid fragment, which is then purified and cloned into an expressionvector. Additional techniques useful in the practice of this inventionmay be found in Current Protocols in Molecular Biology 2007 by JohnWiley and Sons, Inc.; Molecular Cloning: A Laboratory Manual (ThirdEdition) Joseph Sambrook, Peter MacCallum Cancer Institute, Melbourne,Australia; David Russell, University of Texas Southwestern MedicalCenter, Dallas, Cold Spring Harbor.

In embodiments, the immunosuppressants provided herein are coupled tosynthetic nanocarriers. In preferable embodiments, the immunosuppressantis an element that is in addition to the material that makes up thestructure of the synthetic nanocarrier. For example, in one embodiment,where the synthetic nanocarrier is made up of one or more polymers, theimmunosuppressant is a compound that is in addition and coupled to theone or more polymers. As another example, in one embodiment, where thesynthetic nanocarrier is made up of one or more lipids, theimmunosuppressant is again in addition and coupled to the one or morelipids. In embodiments, such as where the material of the syntheticnanocarrier also results in an immunosuppressive (e.g., tolerogenic)effect, the immunosuppressant is an element present in addition to thematerial of the synthetic nanocarrier that results in animmunosuppressive (e.g., tolerogenic) effect.

Other exemplary immunosuppressants include, but are not limited, smallmolecule drugs, natural products, antibodies (e.g., antibodies againstCD20, CD3, CD4), biologics-based drugs, carbohydrate-based drugs,nanoparticles, liposomes, RNAi, antisense nucleic acids, aptamers,methotrexate, NSAIDs; fingolimod; natalizumab; alemtuzumab; anti-CD3;tacrolimus (FK506), etc. Further immunosuppressants, are known to thoseof skill in the art, and the invention is not limited in this respect.

“Inflammatory disease” means any disease, disorder or condition in whichundesired inflammation occurs.

“Load” of the immunosuppressant or antigen is the amount of theimmunosuppressant or antigen coupled to a synthetic nanocarrier based onthe total weight of materials in an entire synthetic nanocarrier(weight/weight). Generally, the load is calculated as an average acrossa population of synthetic nanocarriers. In one embodiment, the load ofthe immunosuppressant on average across the first population ofsynthetic nanocarriers is between 0.0001% and 50%. In anotherembodiment, the load of the antigen on average across the first and/orsecond population of synthetic nanocarriers is between 0.0001% and 50%.In yet another embodiment, the load of the immunosuppressant and/orantigen is between 0.01% and 20%. In a further embodiment, the load ofthe immunosuppressant and/or antigen is between 0.1% and 10%. In still afurther embodiment, the load of the immunosuppressant and/or antigen isbetween 1% and 10%. In yet another embodiment, the load of theimmunosuppressant and/or the antigen is at least 0.1%, at least 0.2%, atleast 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%,at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, atleast 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least9%, at least 10%, at least 11%, at least 12%, at least 13%, at least14%, at least 15%, at least 16%, at least 17%, at least 18%, at least19% or at least 20% on average across a population of syntheticnanocarriers. In yet a further embodiment, the load of theimmunosuppressant and/or the antigen is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% on average across apopulation of synthetic nanocarriers. In some embodiments of the aboveembodiments, the load of the immunosuppressant and/or the antigen is nomore than 25% on average across a population of synthetic nanocarriers.In embodiments, the load is calculated as described in the Examples.

In embodiments of any of the compositions and methods provided, the loadmay be calculated as follows: Approximately 3 mg of syntheticnanocarriers are collected and centrifuged to separate supernatant fromsynthetic nanocarrier pellet. Acetonitrile is added to the pellet, andthe sample is sonicated and centrifuged to remove any insolublematerial. The supernatant and pellet are injected on RP-HPLC andabsorbance is read at 278 nm. The μg found in the pellet is used tocalculate % entrapped (load), μg in supernatant and pellet are used tocalculate total μg recovered.

“Maintenance dose” refers to a dose that is administered to a subject,after an initial dose has resulted in an immunosuppressive (e.g.,tolerogenic) response in a subject, to sustain a desiredimmunosuppressive (e.g., tolerogenic) response. A maintenance dose, forexample, can be one that maintains the tolerogenic effect achieved afterthe initial dose, prevents an undesired immune response in the subject,or prevents the subject becoming a subject at risk of experiencing anundesired immune response, including an undesired level of an immuneresponse. In some embodiments, the maintenance dose is one that issufficient to sustain an appropriate level of a desired immune response.In some embodiments, the maintenance dose is one that is sufficient tosustain an appropriate antibody titre or level of regulatory B cellnumber and/or activity necessary to sustain a tolerogenic immuneresponse or defend against a challenge with an agent that results in anundesired immune response.

“Maximum dimension of a synthetic nanocarrier” means the largestdimension of a nanocarrier measured along any axis of the syntheticnanocarrier. “Minimum dimension of a synthetic nanocarrier” means thesmallest dimension of a synthetic nanocarrier measured along any axis ofthe synthetic nanocarrier. For example, for a spheroidal syntheticnanocarrier, the maximum and minimum dimension of a syntheticnanocarrier would be substantially identical, and would be the size ofits diameter. Similarly, for a cuboidal synthetic nanocarrier, theminimum dimension of a synthetic nanocarrier would be the smallest ofits height, width or length, while the maximum dimension of a syntheticnanocarrier would be the largest of its height, width or length. In anembodiment, a minimum dimension of at least 75%, preferably at least80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in thesample, is equal to or greater than 100 nm. In an embodiment, a maximumdimension of at least 75%, preferably at least 80%, more preferably atleast 90%, of the synthetic nanocarriers in a sample, based on the totalnumber of synthetic nanocarriers in the sample, is equal to or less than5 μm. Preferably, a minimum dimension of at least 75%, preferably atleast 80%, more preferably at least 90%, of the synthetic nanocarriersin a sample, based on the total number of synthetic nanocarriers in thesample, is greater than 110 nm, more preferably greater than 120 nm,more preferably greater than 130 nm, and more preferably still greaterthan 150 nm. Aspects ratios of the maximum and minimum dimensions ofinventive synthetic nanocarriers may vary depending on the embodiment.For instance, aspect ratios of the maximum to minimum dimensions of thesynthetic nanocarriers may vary from 1:1 to 1,000,000:1, preferably from1:1 to 100,000:1, more preferably from 1:1 to 10,000:1, more preferablyfrom 1:1 to 1000:1, still more preferably from 1:1 to 100:1, and yetmore preferably from 1:1 to 10:1. Preferably, a maximum dimension of atleast 75%, preferably at least 80%, more preferably at least 90%, of thesynthetic nanocarriers in a sample, based on the total number ofsynthetic nanocarriers in the sample is equal to or less than 3 μm, morepreferably equal to or less than 2 μm, more preferably equal to or lessthan 1 μm, more preferably equal to or less than 800 nm, more preferablyequal to or less than 600 nm, and more preferably still equal to or lessthan 500 nm. In preferred embodiments, a minimum dimension of at least75%, preferably at least 80%, more preferably at least 90%, of thesynthetic nanocarriers in a sample, based on the total number ofsynthetic nanocarriers in the sample, is equal to or greater than 100nm, more preferably equal to or greater than 120 nm, more preferablyequal to or greater than 130 nm, more preferably equal to or greaterthan 140 nm, and more preferably still equal to or greater than 150 nm.Measurement of synthetic nanocarrier dimensions (e.g., diameter) isobtained by suspending the synthetic nanocarriers in a liquid (usuallyaqueous) media and using dynamic light scattering (DLS) (e.g. using aBrookhaven ZetaPALS instrument). For example, a suspension of syntheticnanocarriers can be diluted from an aqueous buffer into purified waterto achieve a final synthetic nanocarrier suspension concentration ofapproximately 0.01 to 0.1 mg/mL. The diluted suspension may be prepareddirectly inside, or transferred to, a suitable cuvette for DLS analysis.The cuvette may then be placed in the DLS, allowed to equilibrate to thecontrolled temperature, and then scanned for sufficient time to acquirea stable and reproducible distribution based on appropriate inputs forviscosity of the medium and refractive indicies of the sample. Theeffective diameter, or mean of the distribution, is then reported.“Dimension” or “size” or “diameter” of synthetic nanocarriers means themean of a particle size distribution obtained using dynamic lightscattering.

“MHC” refers to major histocompatibility complex, a large genomic regionor gene family found in most vertebrates that encodes MHC molecules thatdisplay fragments or epitopes of processed proteins on the cell surface.The presentation of MHC:peptide on cell surfaces allows for surveillanceby immune cells, usually a T cell. There are two general classes of MHCmolecules: Class I and Class II. Generally, Class I MHC molecules arefound on nucleated cells and present peptides to cytotoxic T cells.Class II MHC molecules are found on certain immune cells, chieflymacrophages, B cells and dendritic cells, collectively known asprofessional APCs. The best-known genes in the MHC region are the subsetthat encodes antigen-presenting proteins on the cell surface. In humans,these genes are referred to as human leukocyte antigen (HLA) genes.

“Non-methoxy-terminated polymer” means a polymer that has at least oneterminus that ends with a moiety other than methoxy. In someembodiments, the polymer has at least two termini that ends with amoiety other than methoxy. In other embodiments, the polymer has notermini that ends with methoxy. “Non-methoxy-terminated, pluronicpolymer” means a polymer other than a linear pluronic polymer withmethoxy at both termini. Polymeric nanoparticles as provided herein cancomprise non-methoxy-terminated polymers or non-methoxy-terminated,pluronic polymers.

“Obtained” means taken directly from a material and used withsubstantially no modification and/or processing.

“Pharmaceutically acceptable excipient” means a pharmacologicallyinactive material used together with the recited synthetic nanocarriersto formulate the inventive compositions. Pharmaceutically acceptableexcipients comprise a variety of materials known in the art, includingbut not limited to saccharides (such as glucose, lactose, and the like),preservatives such as antimicrobial agents, reconstitution aids,colorants, saline (such as phosphate buffered saline), and buffers.

“Protocol” refers to any dosing regimen of one or more substances to asubject. A dosing regimen may include the amount, frequency and/or modeof administration. In some embodiments, such a protocol may be used toadminister one or more compositions of the invention to one or more testsubjects. Immune responses in these test subject can then be assessed todetermine whether or not the protocol was effective in reducing anundesired immune response or generating a desired immune response (e.g.,the promotion of a tolerogenic effect). Any other therapeutic and/orprophylactic effect may also be assessed instead of or in addition tothe aforementioned immune responses. Whether or not a protocol had adesired effect can be determined using any of the methods providedherein or otherwise known in the art. For example, a population of cellsmay be obtained from a subject to which a composition provided hereinhas been administered according to a specific protocol in order todetermine whether or not specific immune cells, cytokines, antibodies,etc. were reduced, generated, activated, etc. Useful methods fordetecting the presence and/or number of immune cells include, but arenot limited to, flow cytometric methods (e.g., FACS) andimmunohistochemistry methods. Antibodies and other binding agents forspecific staining of immune cell markers, are commercially available.Such kits typically include staining reagents for multiple antigens thatallow for FACS-based detection, separation and/or quantitation of adesired cell population from a heterogeneous population of cells.

“Providing a subject” is any action or set of actions that causes aclinician to come in contact with a subject and administer a compositionprovided herein thereto or to perform a method provided hereinthereupon. Preferably, the subject is one who is in need of atolerogenic immune response as provided herein. The action or set ofactions may be either directly oneself or indirectly, such as, but notlimited to, an unrelated third party that takes an action throughreliance on one's words or deeds.

“Regulatory B cells” are art-recognized and refer to a type oftolerogenic B cells or that have suppressive regulatory function.Surface markers and chemokine profiles characteristic of regulatory Bcells, as well as subsets of regulatory B cells (e.g., IL-10 producingBregs) are known to those of skill in the art (e.g., as described inDiLillo et al., B10 cells and regulatory B cells balance immune responseduring inflammation, autoimmunity, and cancer, Ann N.Y. Acad. Sci. 1183(2010) 38-57, ISSN 0077-8923; the entire contents of which areincorporated herein by reference). The presence of regulatory B cellscan be determined by intracellular staining for IL-10 by flow cytometry.For example, after treatment B cells can be stained for surface markers,then fixed and permeabilized and stained for intracellular IL-10 andanalyzed by flow cytometry.

“Subject” means animals, including warm blooded mammals such as humansand primates; avians; domestic household or farm animals such as cats,dogs, sheep, goats, cattle, horses and pigs; laboratory animals such asmice, rats and guinea pigs; fish; reptiles; zoo and wild animals; andthe like.

“Substantially no MHC Class I-restricted epitopes” refers to the absenceof MHC Class I-restricted epitopes in an amount that by itself, withinthe context of the antigen, in conjunction with a carrier or inconjunction with an inventive composition stimulates substantialactivation of an immune response specific to the epitope presented inthe Class I context. In embodiments, a composition with substantially noMHC I-restricted epitopes does not contain a measurable amount of MHCClass I-restricted epitopes of an antigen. In other embodiments, such acomposition may comprise a measurable amount of MHC Class I-restrictedepitopes of an antigen but said amount is not effective to generate ameasurable cytotoxic T cell immune response (by itself, within thecontext of the antigen, in conjunction with a carrier or in conjunctionwith an inventive composition) or is not effective to generate asignificant measurable cytotoxic T cell immune response (by itself,within the context of the antigen, in conjunction with a carrier or inconjunction with an inventive composition). In some embodiments, asignificant measurable cytotoxic T cell immune response is one thatproduces or would be expected to produce an adverse clinical result in asubject. In other embodiments, a significant measurable cytotoxic T cellimmune response is one that is greater than the level of the same typeof immune response produced by a control antigen (e.g., one known not tocomprise MHC Class I-restricted epitopes or to stimulate cytotoxic Tcell immune responses).

In embodiments, the compositions do not comprise MHC Class I-restrictedepitopes (by itself, within the context of the antigen, in conjunctionwith a carrier or in conjunction with an inventive composition) thatgenerate antigen-specific cytotoxic T cell immune responses or anundesired level thereof. In some embodiments, to ensure that acomposition does not comprise such epitopes, antigens are selected suchthat they do not comprise MHC Class I-restricted epitopes for couplingto the synthetic nanocarriers as provided herein. In other embodiments,to ensure that a composition does not comprise such epitopes, syntheticnanocarriers coupled to antigen are produced and tested for cytotoxic Tcell immune responses. The appropriate synthetic nanocarriers may thenbe selected.

“Synthetic nanocarrier(s)” means a discrete object that is not found innature, and that possesses at least one dimension that is less than orequal to 5 microns in size. Albumin nanoparticles are generally includedas synthetic nanocarriers, however in certain embodiments the syntheticnanocarriers do not comprise albumin nanoparticles. In embodiments,inventive synthetic nanocarriers do not comprise chitosan. In otherembodiments, inventive synthetic nanocarriers are not lipid-basednanoparticles. In further embodiments, inventive synthetic nanocarriersdo not comprise a phospholipid.

A synthetic nanocarrier can be, but is not limited to, one or aplurality of lipid-based nanoparticles (also referred to herein as lipidnanoparticles, i.e., nanoparticles where the majority of the materialthat makes up their structure are lipids), polymeric nanoparticles,metallic nanoparticles, surfactant-based emulsions, dendrimers,buckyballs, nanowires, virus-like particles (i.e., particles that areprimarily made up of viral structural proteins but that are notinfectious or have low infectivity), peptide or protein-based particles(also referred to herein as protein particles, i.e., particles where themajority of the material that makes up their structure are peptides orproteins) (such as albumin nanoparticles) and/or nanoparticles that aredeveloped using a combination of nanomaterials such as lipid-polymernanoparticles. Synthetic nanocarriers may be a variety of differentshapes, including but not limited to spheroidal, cuboidal, pyramidal,oblong, cylindrical, toroidal, and the like. Synthetic nanocarriersaccording to the invention comprise one or more surfaces. Exemplarysynthetic nanocarriers that can be adapted for use in the practice ofthe present invention comprise: (1) the biodegradable nanoparticlesdisclosed in U.S. Pat. No. 5,543,158 to Gref et al., (2) the polymericnanoparticles of Published US Patent Application 20060002852 to Saltzmanet al., (3) the lithographically constructed nanoparticles of PublishedUS Patent Application 20090028910 to DeSimone et al., (4) the disclosureof WO 2009/051837 to von Andrian et al., (5) the nanoparticles disclosedin Published US Patent Application 2008/0145441 to Penades et al., (6)the protein nanoparticles disclosed in Published US Patent Application20090226525 to de los Rios et al., (7) the virus-like particlesdisclosed in published US Patent Application 20060222652 to Sebbel etal., (8) the nucleic acid coupled virus-like particles disclosed inpublished US Patent Application 20060251677 to Bachmann et al., (9) thevirus-like particles disclosed in WO2010047839A1 or WO2009106999A2, (10)the nanoprecipitated nanoparticles disclosed in P. Paolicelli et al.,“Surface-modified PLGA-based Nanoparticles that can EfficientlyAssociate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853(2010), or (11) apoptotic cells, apoptotic bodies or the synthetic orsemisynthetic mimics disclosed in U.S. Publication 2002/0086049. Inembodiments, synthetic nanocarriers may possess an aspect ratio greaterthan 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.

Synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface with hydroxyl groups thatactivate complement or alternatively comprise a surface that consistsessentially of moieties that are not hydroxyl groups that activatecomplement. In a preferred embodiment, synthetic nanocarriers accordingto the invention that have a minimum dimension of equal to or less thanabout 100 nm, preferably equal to or less than 100 nm, do not comprise asurface that substantially activates complement or alternativelycomprise a surface that consists essentially of moieties that do notsubstantially activate complement. In a more preferred embodiment,synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface that activates complement oralternatively comprise a surface that consists essentially of moietiesthat do not activate complement. In embodiments, synthetic nanocarriersexclude virus-like particles. In embodiments, synthetic nanocarriers maypossess an aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5,1:7, or greater than 1:10.

“T cell antigen” means a CD4+ T-cell antigen or CD8+ cell antigen. “CD4+T-cell antigen” means any antigen that is recognized by and triggers animmune response in a CD4+ T-cell e.g., an antigen that is specificallyrecognized by a T-cell receptor on a CD4+T cell via presentation of theantigen or portion thereof bound to a Class II major histocompatabilitycomplex molecule (MHC). “CD8+ T cell antigen” means any antigen that isrecognized by and triggers an immune response in a CD8+ T-cell e.g., anantigen that is specifically recognized by a T-cell receptor on a CD8+Tcell via presentation of the antigen or portion thereof bound to a ClassI major histocompatability complex molecule (MHC). In some embodiments,an antigen that is a T cell antigen is also a B cell antigen. In otherembodiments, the T cell antigen is not also a B cell antigen. T cellantigens generally are proteins or peptides.

A “therapeutic protein” refers to any protein or protein-based therapythat may be administered to a subject and have a therapeutic effect.Such therapies include protein replacement and protein supplementationtherapies. Such therapies also include the administration of exogenousor foreign protein, antibody therapies, and cell or cell-basedtherapies. Therapeutic proteins include enzymes, enzyme cofactors,hormones, blood clotting factors, cytokines, growth factors, monoclonalantibodies and polyclonal antibodies. Examples of other therapeuticproteins are provided elsewhere herein. Therapeutic proteins may beproduced in, on or by cells and may be obtained from such cells oradministered in the form of such cells. In embodiments, the therapeuticprotein is produced in, on or by mammalian cells, insect cells, yeastcells, bacteria cells, plant cells, transgenic animal cells, transgenicplant cells, etc. The therapeutic protein may be recombinantly producedin such cells. The therapeutic protein may be produced in, on or by avirally transformed cell. The therapeutic protein may also be producedin, on or by autologous cells that have been transfected, transduced orotherwise manipulated to express it. Alternatively, the therapeuticprotein may be administered as a nucleic acid or by introducing anucleic acid into a virus, VLP, liposome, etc. Alternatively, thetherapeutic protein may be obtained from such forms and administered asthe therapeutic protein itself. Subjects, therefore, include any subjectthat has received, is receiving or will receive any of the foregoing.Such subject includes subjects that have received, is receiving or willreceive gene therapy, autologous cells that have been transfected,transduced or otherwise manipulated to express a therapeutic protein,polypeptide or peptide; or cells that express a therapeutic protein,polypeptide or peptide.

“Therapeutic protein antigen” means an antigen that is associated with atherapeutic protein that can be, or a portion of which can be, presentedfor recognition by cells of the immune system and can generate anundesired immune response (e.g., the production of therapeuticprotein-specific antibodies) against the therapeutic protein.Therapeutic protein antigens generally include proteins, polypeptides,peptides, lipoproteins, or are contained or expressed in, on or bycells.

“Tolerogenic immune response” means any immune response that can lead toimmune suppression specific to an antigen or a cell, tissue, organ, etc.that expresses such an antigen. Such immune responses include anyreduction, delay or inhibition in an undesired immune response specificto the antigen or cell, tissue, organ, etc. that expresses such antigen.Such immune responses also include any stimulation, production,induction, promotion or recruitment in a desired immune responsespecific to the antigen or cell, tissue, organ, etc. that expresses suchantigen. Tolerogenic immune responses, therefore, include the absence ofor reduction in an undesired immune response to an antigen that can bemediated by antigen reactive cells as well as the presence or promotionof suppressive cells. Tolerogenic immune responses as provided hereininclude immunological tolerance. To “generate a tolerogenic immuneresponse” refers to the generation of any of the foregoing immuneresponses specific to an antigen or cell, tissue, organ, etc. thatexpresses such antigen. The tolerogenic immune response can be theresult of MHC Class I-restricted presentation and/or MHC ClassII-restricted presentation and/or B cell presentation and/orpresentation by CD1d, etc. In embodiments, to ensure that a compositioncomprises desired epitopes, antigens are selected such that theycomprise such epitopes for coupling to the synthetic nanocarriers asprovided herein. In other embodiments, to ensure that a compositioncomprises such epitopes, synthetic nanocarriers coupled to an antigenare produced and tested for regulatory B cell immune responses, such asactivation or generation. The appropriate synthetic nanocarriers maythen be selected.

Tolerogenic immune responses include any reduction, delay or inhibitionin CD4+ T cell, CD8+ T cell or B cell proliferation and/or activity.Tolerogenic immune responses also include a reduction inantigen-specific antibody production. Tolerogenic immune responses canalso include any response that leads to the stimulation, induction,production or recruitment of regulatory cells, such as CD4+ Treg cells,CD8+ Treg cells, Breg cells, etc. In some embodiments, the tolerogenicimmune response, is one that results in the conversion to a regulatoryphenotype characterized by the production, induction, stimulation orrecruitment of regulatory cells.

Tolerogenic immune responses also include any response that leads to thestimulation, production or recruitment of CD4+ Treg cells and/or CD8+Treg cells. CD4+ Treg cells can express the transcription factor FoxP3and inhibit inflammatory responses and auto-immune inflammatory diseases(Human regulatory T cells in autoimmune diseases. Cvetanovich G L,Hafler D A. Curr Opin Immunol. 2010 December; 22(6):753-60. Regulatory Tcells and autoimmunity. Vila J, Isaacs J D, Anderson A E. Curr OpinHematol. 2009 July; 16(4):274-9). Such cells also suppress T-cell helpto B-cells and induce tolerance to both self and foreign antigens(Therapeutic approaches to allergy and autoimmunity based on FoxP3+regulatory T-cell activation and expansion. Miyara M, Wing K, SakaguchiS. J Allergy Clin Immunol. 2009 April; 123(4):749-55). CD4+ Treg cellsrecognize antigen when presented by Class II proteins on APCs. CD8+ Tregcells, which recognize antigen presented by Class I (and Qa-1), can alsosuppress T-cell help to B-cells and result in activation ofantigen-specific suppression inducing tolerance to both self and foreignantigens. Disruption of the interaction of Qa-1 with CD8+ Treg cells hasbeen shown to dysregulate immune responses and results in thedevelopment of auto-antibody formation and an auto-immune lethalsystemic-lupus-erythematosus (Kim et al., Nature. 2010 Sep. 16, 467(7313): 328-32). CD8+ Treg cells have also been shown to inhibit modelsof autoimmune inflammatory diseases including rheumatoid arthritis andcolitis (CD4+CD25+ regulatory T cells in autoimmune arthritis. Oh S,Rankin A L, Caton A J. Immunol. Rev. 2010 January; 233(1):97-111.Regulatory T cells in inflammatory bowel disease. Boden E K, Snapper SB. Curr Opin Gastroenterol. 2008 November; 24(6):733-41). In someembodiments, the compositions provided can effectively result in bothtypes of responses (CD4+ Treg and CD8+ Treg). In other embodiments,FoxP3 can be induced in other immune cells, such as macrophages, iNKTcells, etc., and the compositions provided herein can result in one ormore of these responses as well.

Tolerogenic immune responses also include, but are not limited to, theinduction of regulatory cytokines, such as Treg cytokines; induction ofinhibitory cytokines; the inhibition of inflammatory cytokines (e.g.,IL-4, IL-1b, IL-5, TNF-α, IL-6, GM-CSF, IFN-γ, IL-2, IL-9, IL-12, IL-17,IL-18, IL-21, IL-22, IL-23, M-CSF, C reactive protein, acute phaseprotein, chemokines (e.g., MCP-1, RANTES, MIP-1α, MIP-1β, MIG, ITAC orIP-10), the production of anti-inflammatory cytokines (e.g., IL-4,IL-13, IL-10, etc.), chemokines (e.g., CCL-2, CXCL8), proteases (e.g.,MMP-3, MMP-9), leukotrienes (e.g., CysLT-1, CysLT-2), prostaglandins(e.g., PGE2) or histamines; the inhibition of polarization to a Th17,Th1 or Th2 immune response; the inhibition of effector cell-specificcytokines: Th17 (e.g., IL-17, IL-25), Th1 (IFN-γ), Th2 (e.g., IL-4,IL-13); the inhibition of Th1-, Th2- or TH17-specific transcriptionfactors; the inhibition of proliferation of effector T cells; theinduction of apoptosis of effector T cells; the induction of tolerogenicdendritic cell-specific genes, the induction of FoxP3 expression, theinhibition of IgE induction or IgE-mediated immune responses; theinhibition of antibody responses (e.g., antigen-specific antibodyproduction); the inhibition of T helper cell response; the production ofTGF-β and/or IL-10; the inhibition of effector function ofautoantibodies (e.g., inhibition in the depletion of cells, cell ortissue damage or complement activation); etc.

Any of the foregoing may be measured in vivo in one or more animalmodels or may be measured in vitro. One of ordinary skill in the art isfamiliar with such in vivo or in vitro measurements. Undesired immuneresponses or tolerogenic immune responses can be monitored using, forexample, methods of assessing immune cell number and/or function,tetramer analysis, ELISPOT, flow cytometry-based analysis of cytokineexpression, cytokine secretion, cytokine expression profiling, geneexpression profiling, protein expression profiling, analysis of cellsurface markers, PCR-based detection of immune cell receptor gene usage(see T. Clay et al., “Assays for Monitoring Cellular Immune Response toActive Immunotherapy of Cancer” Clinical Cancer Research 7:1127-1135(2001)), etc. Undesired immune responses or tolerogenic immune responsesmay also be monitored using, for example, methods of assessing proteinlevels in plasma or serum, immune cell proliferation and/or functionalassays, etc. In some embodiments, tolerogenic immune responses can bemonitored by assessing the induction of FoxP3. In addition, specificmethods are described in more detail in the Examples.

Preferably, tolerogenic immune responses lead to the inhibition of thedevelopment, progression or pathology of the diseases, disorders orconditions described herein. Whether or not the inventive compositionscan lead to the inhibition of the development, progression or pathologyof the diseases, disorders or conditions described herein can bemeasured with animal models of such diseases, disorders or conditions.In some embodiments, the reduction of an undesired immune response orgeneration of a tolerogenic immune response may be assessed bydetermining clinical endpoints, clinical efficacy, clinical symptoms,disease biomarkers and/or clinical scores. Undesired immune responses ortolerogenic immune responses can also be assessed with diagnostic teststo assess the presence or absence of a disease, disorder or condition asprovided herein. Undesired or desired immune responses can further beassessed by methods of measuring therapeutic proteins levels and/orfunction in a subject. In embodiments, methods for monitoring orassessing undesired allergic responses include assessing an allergicresponse in a subject by skin reactivity and/or allergen-specificantibody production.

In some embodiments, monitoring or assessing the generation of anundesired immune response or a tolerogenic immune response in a subjectcan be prior to the administration of a composition of syntheticnanocarriers provided herein and/or prior to administration of atransplantable graft or therapeutic protein or exposure to an allergen.In other embodiments, assessing the generation of an undesired immuneresponse or tolerogenic immune response can be after administration of acomposition of synthetic nanocarriers provided herein and/or afteradministration of a transplantable graft or therapeutic protein orexposure to an allergen. In some embodiments, the assessment is doneafter administration of the composition of synthetic nanocarriers, butprior to administration of a transplantable graft or therapeutic proteinor exposure to an allergen. In other embodiments, the assessment is doneafter administration of a transplantable graft or therapeutic protein orexposure to an allergen, but prior to administration of the composition.In still other embodiments, the assessment is performed prior to boththe administration of the synthetic nanocarriers and administration of atransplantable graft or therapeutic protein or exposure to an allergen,while in yet other embodiments the assessment is performed after boththe administration of synthetic nanocarriers and the administration of atransplantable graft or therapeutic protein or exposure to an allergen.In further embodiments, the assessment is performed both prior to andafter the administration of the synthetic nanocarriers and/oradministration of a transplantable graft or therapeutic protein orexposure to an allergen. In still other embodiments, the assessment isperformed more than once on the subject to determine that a desirableimmune state is maintained in the subject, such as a subject that has oris at risk of having an inflammatory disease, an autoimmune disease, anallergy, organ or tissue rejection or graft verus host disease. Othersubjects include those that have undergone or will undergotransplantation as well as those that have received, are receiving orwill receive a therapeutic protein against which they have experienced,are experiencing or are expected to experience an undesired immuneresponse.

An antibody response can be assessed by determining one or more antibodytiters. “Antibody titer” means a measurable level of antibodyproduction. Methods for measuring antibody titers are known in the artand include Enzyme-linked Immunosorbent Assay (ELISA). In embodiments,the antibody response can be quantitated, for example, as the number ofantibodies, concentration of antibodies or titer. The values can beabsolute or they can be relative. Assays for quantifying an antibodyresponse include antibody capture assays, enzyme-linked immunosorbentassays (ELISAs), inhibition liquid phase absorption assays (ILPAAs),rocket immunoelectrophoresis (RIE) assays and line immunoelectrophoresis(LIE) assays. When an antibody response is compared to another antibodyresponse the same type of quantitative value (e.g., titer) and method ofmeasurement (e.g., ELISA) is preferably used to make the comparison.

An ELISA method for measuring an antibody titer, for example, a typicalsandwich ELISA, may consist of the following steps (i) preparing anELISA-plate coating material such that the antibody target of interestis coupled to a substrate polymer or other suitable material (ii)preparing the coating material in an aqueous solution (such as PBS) anddelivering the coating material solution to the wells of a multiwellplate for overnight deposition of the coating onto the multiwell plate(iii) thoroughly washing the multiwell plate with wash buffer (such as0.05% Tween-20 in PBS) to remove excess coating material (iv) blockingthe plate for nonspecific binding by applying a diluent solution (suchas 10% fetal bovine serum in PBS), (v) washing the blocking/diluentsolution from the plate with wash buffer (vi) diluting the serumsample(s) containing antibodies and appropriate standards (positivecontrols) with diluent as required to obtain a concentration thatsuitably saturates the ELISA response (vii) serially diluting the plasmasamples on the multiwell plate such to cover a range of concentrationssuitable for generating an ELISA response curve (viii) incubating theplate to provide for antibody-target binding (ix) washing the plate withwash buffer to remove antibodies not bound to antigen (x) adding anappropriate concentration of a secondary detection antibody in samediluent such as a biotin-coupled detection antibody capable of bindingthe primary antibody (xi) incubating the plate with the applieddetection antibody, followed by washing with wash buffer (xii) adding anenzyme such as streptavidin-HRP (horse radish peroxidase) that will bindto biotin found on biotinylated antibodies and incubating (xiii) washingthe multiwell plate (xiv) adding substrate(s) (such as TMB solution) tothe plate (xv) applying a stop solution (such as 2N sulfuric acid) whencolor development is complete (xvi) reading optical density of the platewells at a specific wavelength for the substrate (450 nm withsubtraction of readings at 570 nm) (xvi) applying a suitablemultiparameter curve fit to the data and defining half-maximal effectiveconcentration (EC50) as the concentration on the curve at which half themaximum OD value for the plate standards is achieved.

A “transplantable graft” refers to a biological material, such as cells,tissues and organs (in whole or in part) that can be administered to asubject. Transplantable grafts may be autografts, allografts, orxenografts of, for example, a biological material such as an organ,tissue, skin, bone, nerves, tendon, neurons, blood vessels, fat, cornea,pluripotent cells, differentiated cells (obtained or derived in vivo orin vitro), etc. In some embodiments, a transplantable graft is formed,for example, from cartilage, bone, extracellular matrix, or collagenmatrices. Transplantable grafts may also be single cells, suspensions ofcells and cells in tissues and organs that can be transplanted.Transplantable cells typically have a therapeutic function, for example,a function that is lacking or diminished in a recipient subject. Somenon-limiting examples of transplantable cells are β-cells, hepatocytes,hematopoietic stem cells, neuronal stem cells, neurons, glial cells, ormyelinating cells. Transplantable cells can be cells that areunmodified, for example, cells obtained from a donor subject and usablein transplantation without any genetic or epigenetic modifications. Inother embodiments, transplantable cells can be modified cells, forexample, cells obtained from a subject having a genetic defect, in whichthe genetic defect has been corrected, or cells that are derived fromreprogrammed cells, for example, differentiated cells derived from cellsobtained from a subject.

“Transplantation” refers to the process of transferring (moving) atransplantable graft into a recipient subject (e.g., from a donorsubject, from an in vitro source (e.g., differentiated autologous orheterologous native or induced pluripotent cells)) and/or from onebodily location to another bodily location in the same subject.

“Undesired immune response” refers to any undesired immune response thatresults from exposure to an antigen, promotes or exacerbates a disease,disorder or condition provided herein (or a symptom thereof), or issymptomatic of a disease, disorder or condition provided herein. Suchimmune responses generally have a negative impact on a subject's healthor is symptomatic of a negative impact on a subject's health.

C. Inventive Compositions

Provided herein are methods and compositions for generating regulatory Bcells. Regulatory B cells are believed to modulate immune responses, forexample, in that IL-10 release from regulatory B cells exertsanti-inflammatory and immunosuppressive effects on most hematopoieticcells. IL-10 also suppresses pro-inflammatory cytokine production bymonocytes and macrophages and the proliferation of antigen-specific CD4+T cells. Without wishing to be bound by any particular theory, it isbelieved that the compositions provided herein can affect the numberand/or activity of regulatory B cells in vitro and/or in vivo by, forexample, interacting with naïve B cells or regulatory B cell precursors,resulting in an induction of regulatory B cell maturation or an increasein regulatory B cell proliferation and/or activity. The effect onregulatory B cell number and/or activity may also be a result of thegeneration of other regulatory cell or tolerogenic immune responses,such as through regulatory T cell production and/or activation.

Provided herein are tolerogenic methods that include the administrationof synthetic nanocarrier compositions comprising immunosuppressants andB cell and/or MHC Class II-restricted epitopes of an antigen and relatedcompositions. Such methods and compositions are useful for generating,stimulating or recruiting regulatory B cells and promoting thegeneration of tolerogenic immune responses. The methods provided mayalso include the administration therapeutic proteins and/ortransplantable grafts. The compositions provided, therefore, may alsoinclude the therapeutic proteins and/or transplantable grafts. Thecompositions may be administered to subjects in which a tolerogenicimmune response is desired. Such subjects include those that have or areat risk of having an inflammatory disease, an autoimmune disease, anallergy, organ or tissue rejection or graft versus host disease. Suchsubjects also include those that have been, are being or will beadministered a therapeutic protein against which the subject hasexperienced or is expected to experience an undesired immune response.Such subjects also include those that have undergone or will undergotransplantation.

Preferably, the compositions of the invention result in the generation,recruitment or activation of regulatory B cells. One hallmark ofregulatory B cells is the ability to produce and secrete IL-10. Someregulatory B cells are CD1d+ or CD1dhigh. Some regulatory B cells areCD5+ and/or CD19+. For example, some regulatory B cells areCD1dhighCD5+CD19+. Some regulatory B cells are CD24+ or CD24high; and/orCD38+ or CD38high. For example, some regulatory B cells areCD19+CD24highCD38high. Some regulatory B cells produce and secreteIL-10. Additional surface markers and chemokine secretion profiles thatcan be used to identify regulatory B cells are known to those of skillin the art. Based on the knowledge of surface markers useful for theidentification of various regulatory B cell populations, those of skillin the art are able to identify and enumerate regulatory B cells in aheterogeneous population of cells, for example, in a population of cellsin culture or in a population of cells obtained from a subject.

As mentioned above, the synthetic nanocarriers are designed to compriseB cell eptiopes and/or MHC Class II-restricted epitopes and/orimmunosuppressants. A wide variety of synthetic nanocarriers can be usedaccording to the invention. In some embodiments, synthetic nanocarriersare spheres or spheroids. In some embodiments, synthetic nanocarriersare flat or plate-shaped. In some embodiments, synthetic nanocarriersare cubes or cubic. In some embodiments, synthetic nanocarriers areovals or ellipses. In some embodiments, synthetic nanocarriers arecylinders, cones, or pyramids.

In some embodiments, it is desirable to use a population of syntheticnanocarriers that is relatively uniform in terms of size, shape, and/orcomposition so that each synthetic nanocarrier has similar properties.For example, at least 80%, at least 90%, or at least 95% of thesynthetic nanocarriers, based on the total number of syntheticnanocarriers, may have a minimum dimension or maximum dimension thatfalls within 5%, 10%, or 20% of the average diameter or averagedimension of the synthetic nanocarriers. In some embodiments, apopulation of synthetic nanocarriers may be heterogeneous with respectto size, shape, and/or composition.

Synthetic nanocarriers can be solid or hollow and can comprise one ormore layers. In some embodiments, each layer has a unique compositionand unique properties relative to the other layer(s). To give but oneexample, synthetic nanocarriers may have a core/shell structure, whereinthe core is one layer (e.g. a polymeric core) and the shell is a secondlayer (e.g. a lipid bilayer or monolayer). Synthetic nanocarriers maycomprise a plurality of different layers.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more lipids. In some embodiments, a synthetic nanocarrier maycomprise a liposome. In some embodiments, a synthetic nanocarrier maycomprise a lipid bilayer. In some embodiments, a synthetic nanocarriermay comprise a lipid monolayer. In some embodiments, a syntheticnanocarrier may comprise a micelle. In some embodiments, a syntheticnanocarrier may comprise a core comprising a polymeric matrix surroundedby a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.). In someembodiments, a synthetic nanocarrier may comprise a non-polymeric core(e.g., metal particle, quantum dot, ceramic particle, bone particle,viral particle, proteins, nucleic acids, carbohydrates, etc.) surroundedby a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.).

In other embodiments, synthetic nanocarriers may comprise metalparticles, quantum dots, ceramic particles, etc. In some embodiments, anon-polymeric synthetic nanocarrier is an aggregate of non-polymericcomponents, such as an aggregate of metal atoms (e.g., gold atoms).

In some embodiments, synthetic nanocarriers may optionally comprise oneor more amphiphilic entities. In some embodiments, an amphiphilic entitycan promote the production of synthetic nanocarriers with increasedstability, improved uniformity, or increased viscosity. In someembodiments, amphiphilic entities can be associated with the interiorsurface of a lipid membrane (e.g., lipid bilayer, lipid monolayer,etc.). Many amphiphilic entities known in the art are suitable for usein making synthetic nanocarriers in accordance with the presentinvention. Such amphiphilic entities include, but are not limited to,phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine(DPPC); dioleylphosphatidyl ethanolamine (DOPE);dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine;cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate;diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such aspolyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surfaceactive fatty acid, such as palmitic acid or oleic acid; fatty acids;fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides;sorbitan trioleate (Span®85) glycocholate; sorbitan monolaurate(Span®20); polysorbate 20 (Tween®20); polysorbate 60 (Tween®60);polysorbate 65 (Tween®65); polysorbate 80 (Tween®80); polysorbate 85(Tween®85); polyoxyethylene monostearate; surfactin; a poloxomer; asorbitan fatty acid ester such as sorbitan trioleate; lecithin;lysolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin;phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid;cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol;stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerolricinoleate; hexadecyl sterate; isopropyl myristate; tyloxapol;poly(ethylene glycol)5000-phosphatidylethanolamine; poly(ethyleneglycol)400-monostearate; phospholipids; synthetic and/or naturaldetergents having high surfactant properties; deoxycholates;cyclodextrins; chaotropic salts; ion pairing agents; and combinationsthereof. An amphiphilic entity component may be a mixture of differentamphiphilic entities. Those skilled in the art will recognize that thisis an exemplary, not comprehensive, list of substances with surfactantactivity. Any amphiphilic entity may be used in the production ofsynthetic nanocarriers to be used in accordance with the presentinvention.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more carbohydrates. Carbohydrates may be natural or synthetic. Acarbohydrate may be a derivatized natural carbohydrate. In certainembodiments, a carbohydrate comprises monosaccharide or disaccharide,including but not limited to glucose, fructose, galactose, ribose,lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose,arabinose, glucoronic acid, galactoronic acid, mannuronic acid,glucosamine, galatosamine, and neuramic acid. In certain embodiments, acarbohydrate is a polysaccharide, including but not limited to pullulan,cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose(HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran,cyclodextran, glycogen, hydroxyethylstarch, carageenan, glycon, amylose,chitosan, N,O-carboxylmethylchitosan, algin and alginic acid, starch,chitin, inulin, konjac, glucommannan, pustulan, heparin, hyaluronicacid, curdlan, and xanthan. In embodiments, the inventive syntheticnanocarriers do not comprise (or specifically exclude) carbohydrates,such as a polysaccharide. In certain embodiments, the carbohydrate maycomprise a carbohydrate derivative such as a sugar alcohol, includingbut not limited to mannitol, sorbitol, xylitol, erythritol, maltitol,and lactitol.

In some embodiments, synthetic nanocarriers can comprise one or morepolymers. In some embodiments, the synthetic nanocarriers comprise oneor more polymers that is a nonmethoxy-terminated, pluronic polymer. Insome embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or99% (weight/weight) of the polymers that make up the syntheticnanocarriers are non-methoxy-terminated, pluronic polymers. In someembodiments, all of the polymers that make up the synthetic nanocarriersare non-methoxy-terminated, pluronic polymers. In some embodiments, thesynthetic nanocarriers can comprise one or more polymers that is anon-methoxy-terminated polymer. In some embodiments, at least 1%, 2%,3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight) of thepolymers that make up the synthetic nanocarriers arenonmethoxy-terminated polymers. In some embodiments, all of the polymersthat make up the synthetic nanocarriers are non-methoxy-terminatedpolymers. In some embodiments, the synthetic nanocarriers comprise oneor more polymers that do not comprise pluronic polymer. In someembodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99%(weight/weight) of the polymers that make up the synthetic nanocarriersdo not comprise pluronic polymer. In some embodiments, all of thepolymers that make up the synthetic nanocarriers do not comprisepluronic polymer. In some embodiments, such a polymer can be surroundedby a coating layer (e.g., liposome, lipid monolayer, micelle, etc.). Insome embodiments, various elements of the synthetic nanocarriers can becoupled with the polymer.

The immunosuppressants and/or antigens can be coupled to the syntheticnanocarriers by any of a number of methods. Generally, the coupling canbe a result of bonding between the immunosuppressants and/or antigensand the synthetic nanocarriers. This bonding can result in theimmunosuppressants and/or antigens being attached to the surface of thesynthetic nanocarrierss and/or contained within (encapsulated) thesynthetic nanocarriers. In some embodiments, however, theimmunosuppressants and/or antigens are encapsulated by the syntheticnanocarriers as a result of the structure of the synthetic nanocarriersrather than bonding to the synthetic nanocarriers. In preferableembodiments, the synthetic nanocarriers comprise a polymer as providedherein, and the immunosuppressants and/or antigens are coupled to thepolymer.

When coupling occurs as a result of bonding between theimmunosuppressants and/or antigens and synthetic nanocarriers, thecoupling may occur via a coupling moiety. A coupling moiety can be anymoiety through which an immunosuppressant and/or antigen is bonded to asynthetic nanocarrier. Such moieties include covalent bonds, such as anamide bond or ester bond, as well as separate molecules that bond(covalently or non-covalently) the immunosuppressant and/or antigen tothe synthetic nanocarrier. Such molecules include linkers or polymers ora unit thereof. For example, the coupling moiety can comprise a chargedpolymer to which an immunosuppressant and/or antigen electrostaticallybinds. As another example, the coupling moiety can comprise a polymer orunit thereof to which it is covalently bonded.

In preferred embodiments, the synthetic nanocarriers comprise a polymeras provided herein. These synthetic nanocarriers can be completelypolymeric or they can be a mix of polymers and other materials.

In some embodiments, the polymers of a synthetic nanocarrier associateto form a polymeric matrix. In some of these embodiments, a component,such as an immunosuppressant or antigen, can be covalently associatedwith one or more polymers of the polymeric matrix. In some embodiments,covalent association is mediated by a linker. In some embodiments, acomponent can be noncovalently associated with one or more polymers ofthe polymeric matrix. For example, in some embodiments a component canbe encapsulated within, surrounded by, and/or dispersed throughout apolymeric matrix. Alternatively or additionally, a component can beassociated with one or more polymers of a polymeric matrix byhydrophobic interactions, charge interactions, van der Waals forces,etc. A wide variety of polymers and methods for forming polymericmatrices therefrom are known conventionally.

Polymers may be natural or unnatural (synthetic) polymers. Polymers maybe homopolymers or copolymers comprising two or more monomers. In termsof sequence, copolymers may be random, block, or comprise a combinationof random and block sequences. Typically, polymers in accordance withthe present invention are organic polymers.

In some embodiments, the polymer comprises a polyester, polycarbonate,polyamide, or polyether, or unit thereof. In other embodiments, thepolymer comprises poly(ethylene glycol) (PEG), polypropylene glycol,poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid),or a polycaprolactone, or unit thereof. In some embodiments, it ispreferred that the polymer is biodegradable. Therefore, in theseembodiments, it is preferred that if the polymer comprises a polyether,such as poly(ethylene glycol) or polypropylene glycol or unit thereof,the polymer comprises a block-co-polymer of a polyether and abiodegradable polymer such that the polymer is biodegradable. In otherembodiments, the polymer does not solely comprise a polyether or unitthereof, such as poly(ethylene glycol) or polypropylene glycol or unitthereof.

Other examples of polymers suitable for use in the present inventioninclude, but are not limited to polyethylenes, polycarbonates (e.g.poly(1,3-dioxan-2one)), polyanhydrides (e.g. poly(sebacic anhydride)),polypropylfumerates, polyamides (e.g. polycaprolactam), polyacetals,polyethers, polyesters (e.g., polylactide, polyglycolide,polylactide-co-glycolide, polycaprolactone, polyhydroxyacid (e.g.poly(β-hydroxyalkanoate))), poly(orthoesters), polycyanoacrylates,polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polyureas, polystyrenes, and polyamines, polylysine,polylysine-PEG copolymers, and poly(ethyleneimine), poly(ethyleneimine)-PEG copolymers.

In some embodiments, polymers in accordance with the present inventioninclude polymers which have been approved for use in humans by the U.S.Food and Drug Administration (FDA) under 21 C.F.R. §177.2600, includingbut not limited to polyesters (e.g., polylactic acid,poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone,poly(1,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride));polyethers (e.g., polyethylene glycol); polyurethanes;polymethacrylates; polyacrylates; and polycyanoacrylates.

In some embodiments, polymers can be hydrophilic. For example, polymersmay comprise anionic groups (e.g., phosphate group, sulphate group,carboxylate group); cationic groups (e.g., quaternary amine group); orpolar groups (e.g., hydroxyl group, thiol group, amine group). In someembodiments, a synthetic nanocarrier comprising a hydrophilic polymericmatrix generates a hydrophilic environment within the syntheticnanocarrier. In some embodiments, polymers can be hydrophobic. In someembodiments, a synthetic nanocarrier comprising a hydrophobic polymericmatrix generates a hydrophobic environment within the syntheticnanocarrier. Selection of the hydrophilicity or hydrophobicity of thepolymer may have an impact on the nature of materials that areincorporated (e.g. coupled) within the synthetic nanocarrier.

In some embodiments, polymers may be modified with one or more moietiesand/or functional groups. A variety of moieties or functional groups canbe used in accordance with the present invention. In some embodiments,polymers may be modified with polyethylene glycol (PEG), with acarbohydrate, and/or with acyclic polyacetals derived frompolysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). Certainembodiments may be made using the general teachings of U.S. Pat. No.5,543,158 to Gref et al., or WO publication WO2009/051837 by Von Andrianet al.

In some embodiments, polymers may be modified with a lipid or fatty acidgroup. In some embodiments, a fatty acid group may be one or more ofbutyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEG copolymers and copolymers oflactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers,PLGA-PEG copolymers, and derivatives thereof. In some embodiments,polyesters include, for example, poly(caprolactone),poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine),poly(serine ester), poly(4-hydroxy-L-proline ester),poly[α-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA are characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid:glycolic acid ratio. In some embodiments, PLGA to be used inaccordance with the present invention is characterized by a lacticacid:glycolic acid ratio of approximately 85:15, approximately 75:25,approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate,poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkylmethacrylate copolymer, glycidyl methacrylate copolymers,polycyanoacrylates, and combinations comprising one or more of theforegoing polymers. The acrylic polymer may comprise fully-polymerizedcopolymers of acrylic and methacrylic acid esters with a low content ofquaternary ammonium groups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g. DNA, or derivatives thereof).Amine-containing polymers such as poly(lysine) (Zauner et al., 1998,Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, BioconjugateChem., 6:7), poly(ethylene imine) (PEI; Boussif et al., 1995, Proc.Natl. Acad. Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers(Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897;Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993,Bioconjugate Chem., 4:372) are positively-charged at physiological pH,form ion pairs with nucleic acids, and mediate transfection in a varietyof cell lines. In embodiments, the inventive synthetic nanocarriers maynot comprise (or may exclude) cationic polymers.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains (Putnam et al., 1999, Macromolecules, 32:3658;Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon et al., 1989,Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121:5633;and Zhou et al., 1990, Macromolecules, 23:3399). Examples of thesepolyesters include poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J.Am. Chem. Soc., 115:11010), poly(serine ester) (Zhou et al., 1990,Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam etal., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem.Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam et al.,1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc.,121:5633).

The properties of these and other polymers and methods for preparingthem are well known in the art (see, for example, U.S. Pat. Nos.6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148;5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665;5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al.,2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc.,123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J.Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181).More generally, a variety of methods for synthesizing certain suitablepolymers are described in Concise Encyclopedia of Polymer Science andPolymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press,1980; Principles of Polymerization by Odian, John Wiley & Sons, FourthEdition, 2004; Contemporary Polymer Chemistry by Allcock et al.,Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in U.S.Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

In some embodiments, polymers can be linear or branched polymers. Insome embodiments, polymers can be dendrimers. In some embodiments,polymers can be substantially cross-linked to one another. In someembodiments, polymers can be substantially free of cross-links. In someembodiments, polymers can be used in accordance with the presentinvention without undergoing a cross-linking step. It is further to beunderstood that inventive synthetic nanocarriers may comprise blockcopolymers, graft copolymers, blends, mixtures, and/or adducts of any ofthe foregoing and other polymers. Those skilled in the art willrecognize that the polymers listed herein represent an exemplary, notcomprehensive, list of polymers that can be of use in accordance withthe present invention.

Compositions according to the invention comprise synthetic nanocarriersin combination with pharmaceutically acceptable excipients, such aspreservatives, buffers, saline, or phosphate buffered saline. Thecompositions may be made using conventional pharmaceutical manufacturingand compounding techniques to arrive at useful dosage forms. In anembodiment, inventive synthetic nanocarriers are suspended in sterilesaline solution for injection together with a preservative.

In embodiments, when preparing synthetic nanocarriers as carriers,methods for coupling components to the synthetic nanocarriers may beuseful. If the component is a small molecule it may be of advantage toattach the component to a polymer prior to the assembly of the syntheticnanocarriers. In embodiments, it may also be an advantage to prepare thesynthetic nanocarriers with surface groups that are used to couple thecomponent to the synthetic nanocarrier through the use of these surfacegroups rather than attaching the component to a polymer and then usingthis polymer conjugate in the construction of synthetic nanocarriers.

In certain embodiments, the coupling can be a covalent linker. Inembodiments, peptides according to the invention can be covalentlycoupled to the external surface via a 1,2,3-triazole linker formed bythe 1,3-dipolar cycloaddition reaction of azido groups on the surface ofthe nanocarrier with antigen or immunosuppressant containing an alkynegroup or by the 1,3-dipolar cycloaddition reaction of alkynes on thesurface of the nanocarrier with components containing an azido group.Such cycloaddition reactions are preferably performed in the presence ofa Cu(I) catalyst along with a suitable Cu(I)-ligand and a reducing agentto reduce Cu(II) compound to catalytic active Cu(I) compound. ThisCu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) can also be referredas the click reaction.

Additionally, the covalent coupling may comprise a covalent linker thatcomprises an amide linker, a disulfide linker, a thioether linker, ahydrazone linker, a hydrazide linker, an imine or oxime linker, an ureaor thiourea linker, an amidine linker, an amine linker, and asulfonamide linker.

An amide linker is formed via an amide bond between an amine on onecomponent with the carboxylic acid group of a second component such asthe nanocarrier. The amide bond in the linker can be made using any ofthe conventional amide bond forming reactions with suitably protectedamino acids or components and activated carboxylic acid suchN-hydroxysuccinimide-activated ester.

A disulfide linker is made via the formation of a disulfide (S—S) bondbetween two sulfur atoms of the form, for instance, of R1-S—S—R2. Adisulfide bond can be formed by thiol exchange of a component containingthiol/mercaptan group (—SH) with another activated thiol group on apolymer or nanocarrier or a nanocarrier containing thiol/mercaptangroups with a component containing activated thiol group.

A triazole linker, specifically a 1,2,3-triazole of the form

wherein R1 and R2 may be any chemical entities, is made by the1,3-dipolar cycloaddition reaction of an azide attached to a firstcomponent such as the nanocarrier with a terminal alkyne attached to asecond component such as the immunosuppressant or antigen. The1,3-dipolar cycloaddition reaction is performed with or without acatalyst, preferably with Cu(I)-catalyst, which links the two componentsthrough a 1,2,3-triazole function. This chemistry is described in detailby Sharpless et al., Angew. Chem. Int. Ed. 41(14), 2596, (2002) andMeldal, et al, Chem. Rev., 2008, 108(8), 2952-3015 and is often referredto as a “click” reaction or CuAAC.

In embodiments, a polymer containing an azide or alkyne group, terminalto the polymer chain is prepared. This polymer is then used to prepare asynthetic nanocarrier in such a manner that a plurality of the alkyne orazide groups are positioned on the surface of that nanocarrier.Alternatively, the synthetic nanocarrier can be prepared by anotherroute, and subsequently functionalized with alkyne or azide groups. Thecomponent is prepared with the presence of either an alkyne (if thepolymer contains an azide) or an azide (if the polymer contains analkyne) group. The component is then allowed to react with thenanocarrier via the 1,3-dipolar cycloaddition reaction with or without acatalyst which covalently couples the component to the particle throughthe 1,4-disubstituted 1,2,3-triazole linker.

A thioether linker is made by the formation of a sulfur-carbon(thioether) bond in the form, for instance, of R1-S—R2. Thioether can bemade by either alkylation of a thiol/mercaptan (—SH) group on onecomponent such as the component with an alkylating group such as halideor epoxide on a second component such as the nanocarrier. Thioetherlinkers can also be formed by Michael addition of a thiol/mercaptangroup on one component to an electron-deficient alkene group on a secondcomponent such as a polymer containing a maleimide group or vinylsulfone group as the Michael acceptor. In another way, thioether linkerscan be prepared by the radical thiol-ene reaction of a thiol/mercaptangroup on one component with an alkene group on a second component suchas a polymer or nanocarrier.

A hydrazone linker is made by the reaction of a hydrazide group on onecomponent with an aldehyde/ketone group on the second component such asthe nanocarrier.

A hydrazide linker is formed by the reaction of a hydrazine group on onecomponent with a carboxylic acid group on the second component such asthe nanocarrier. Such reaction is generally performed using chemistrysimilar to the formation of amide bond where the carboxylic acid isactivated with an activating reagent.

An imine or oxime linker is formed by the reaction of an amine orN-alkoxyamine (or aminooxy) group on one component with an aldehyde orketone group on the second component such as the nanocarrier.

An urea or thiourea linker is prepared by the reaction of an amine groupon one component with an isocyanate or thioisocyanate group on thesecond component such as the nanocarrier.

An amidine linker is prepared by the reaction of an amine group on onecomponent with an imidoester group on the second component such as thenanocarrier.

An amine linker is made by the alkylation reaction of an amine group onone component with an alkylating group such as halide, epoxide, orsulfonate ester group on the second component such as the nanocarrier.Alternatively, an amine linker can also be made by reductive aminationof an amine group on one component with an aldehyde or ketone group onthe second component such as the nanocarrier with a suitable reducingreagent such as sodium cyanoborohydride or sodium triacetoxyborohydride.

A sulfonamide linker is made by the reaction of an amine group on onecomponent with a sulfonyl halide (such as sulfonyl chloride) group onthe second component such as the nanocarrier.

A sulfone linker is made by Michael addition of a nucleophile to a vinylsulfone. Either the vinyl sulfone or the nucleophile may be on thesurface of the nanocarrier or attached to a component.

The component can also be conjugated to the nanocarrier via non-covalentconjugation methods. For example, a negative charged antigen orimmunosuppressant can be conjugated to a positive charged nanocarrierthrough electrostatic adsorption. A component containing a metal ligandcan also be conjugated to a nanocarrier containing a metal complex via ametal-ligand complex.

In embodiments, the component can be attached to a polymer, for examplepolylactic acid-block-polyethylene glycol, prior to the assembly of thesynthetic nanocarrier or the synthetic nanocarrier can be formed withreactive or activatible groups on its surface. In the latter case, thecomponent may be prepared with a group which is compatible with theattachment chemistry that is presented by the synthetic nanocarriers'surface. In other embodiments, a peptide component can be attached toVLPs or liposomes using a suitable linker. A linker is a compound orreagent that capable of coupling two molecules together. In anembodiment, the linker can be a homobifuntional or heterobifunctionalreagent as described in Hermanson 2008. For example, an VLP or liposomesynthetic nanocarrier containing a carboxylic group on the surface canbe treated with a homobifunctional linker, adipic dihydrazide (ADH), inthe presence of EDC to form the corresponding synthetic nanocarrier withthe ADH linker. The resulting ADH linked synthetic nanocarrier is thenconjugated with a peptide component containing an acid group via theother end of the ADH linker on NC to produce the corresponding VLP orliposome peptide conjugate.

For detailed descriptions of available conjugation methods, seeHermanson G T “Bioconjugate Techniques”, 2nd Edition Published byAcademic Press, Inc., 2008. In addition to covalent attachment thecomponent can be coupled by adsorption to a pre-formed syntheticnanocarrier or it can be coupled by encapsulation during the formationof the synthetic nanocarrier.

Any immunosuppressant as provided herein can be coupled to the syntheticnanocarrier. Immunosuppressants include, but are not limited to,statins; mTOR inhibitors, such as rapamycin or a rapamycin analog; TGF-βsignaling agents; TGF-β receptor agonists; histone deacetylase (HDAC)inhibitors; corticosteroids; inhibitors of mitochondrial function, suchas rotenone; P38 inhibitors; NF-κβ inhibitors; adenosine receptoragonists; prostaglandin E2 agonists; phosphodiesterase inhibitors, suchas phosphodiesterase 4 inhibitor; proteasome inhibitors; kinaseinhibitors; G-protein coupled receptor agonists; G-protein coupledreceptor antagonists; glucocorticoids; retinoids; cytokine inhibitors;cytokine receptor inhibitors; cytokine receptor activators; peroxisomeproliferator-activated receptor antagonists; peroxisomeproliferator-activated receptor agonists; histone deacetylaseinhibitors; calcineurin inhibitors; phosphatase inhibitors and oxidizedATPs. Immunosuppressants also include IDO, vitamin D3, cyclosporine A,aryl hydrocarbon receptor inhibitors, resveratrol, azathiopurine,6-mercaptopurine, aspirin, niflumic acid, estriol, tripolide,interleukins (e.g., IL-1, IL-10), cyclosporine A, siRNAs targetingcytokines or cytokine receptors and the like.

Examples of statins include atorvastatin (LIPITOR®, TORVAST®),cerivastatin, fluvastatin (LESCOL®, LESCOL® XL), lovastatin (MEVACOR®,ALTOCOR®, ALTOPREV®), mevastatin (COMPACTIN®), pitavastatin (LIVALO®,PIAVA®), rosuvastatin (PRAVACHOL®, SELEKTINE®, LIPOSTAT®), rosuvastatin(CRESTOR®), and simvastatin (ZOCOR®, LIPEX®).

Examples of mTOR inhibitors include rapamycin and analogs thereof (e.g.,CCL-779, RAD001, AP23573, C20-methallylrapamycin (C20-Marap),C16-(S)-butylsulfonamidorapamycin (C16-BSrap),C16-(S)-3-methylindolerapamycin (C16-iRap) (Bayle et al. Chemistry &Biology 2006, 13:99-107)), AZD8055, BEZ235 (NVP-BEZ235), chrysophanicacid (chrysophanol), deforolimus (MK-8669), everolimus (RAD0001),KU-0063794, PI-103, PP242, temsirolimus, and WYE-354 (available fromSelleck, Houston, Tex., USA).

Examples of TGF-β signaling agents include TGF-β ligands (e.g., activinA, GDF1, GDF11, bone morphogenic proteins, nodal, TGF-βs) and theirreceptors (e.g., ACVR1B, ACVR1c, ACVR2A, ACVR2B, BMPR2, BMPR1A, BMPR1B,TGFβR1, TGFβRII), R-SMADS/co-SMADS (e.g., SMAD1, SMAD2, SMAD3, SMAD4,SMAD5, SMAD8), and ligand inhibitors (e.g, follistatin, noggin, chordin,DAN, lefty, LTBP1, THBS1, Decorin).

Examples of inhibitors of mitochondrial function include atractyloside(dipotassium salt), bongkrekic acid (triammonium salt), carbonyl cyanidem-chlorophenylhydrazone, carboxyatractyloside (e.g., from Atractylisgummifera), CGP-37157, (−)-Deguelin (e.g., from Mundulea sericea), F16,hexokinase II VDAC binding domain peptide, oligomycin, rotenone, Ru360,SFK1, and valinomycin (e.g., from Streptomyces fulvissimus)(EMD4Biosciences, USA).

Examples of P38 inhibitors include SB-203580(4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole),SB-239063(trans-1-(4hydroxycyclohexyl)-4-(fluorophenyl)-5-(2-methoxy-pyrimidin-4-yl)imidazole),SB-220025(5-(2-amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole)),and ARRY-797.

Examples of NF (e.g., NK-κβ inhibitors includeIFRD1,2-(1,8-naphthyridin-2-yl)-Phenol, 5-aminosalicylic acid, BAY11-7082, BAY 11-7085, CAPE (Caffeic Acid Phenethylester),diethylmaleate, IKK-2 Inhibitor IV, IMD 0354, lactacystin, MG-132[Z-Leu-Leu-Leu-CHO], NFκB Activation Inhibitor III, NF-κB ActivationInhibitor II, JSH-23, parthenolide, Phenylarsine Oxide (PAO), PPM-18,pyrrolidinedithiocarbamic acid ammonium salt, QNZ, RO 106-9920,rocaglamide, rocaglamide AL, rocaglamide C, rocaglamide I, rocaglamideJ, rocaglaol, (R)-MG-132, sodium salicylate, triptolide (PG490),wedelolactone.

Examples of adenosine receptor agonists include CGS-21680 and ATL-146e.

Examples of prostaglandin E2 agonists include E-Prostanoid 2 andE-Prostanoid 4.

Examples of phosphodiesterase inhibitors (non-selective and selectiveinhibitors) include caffeine, aminophylline, IBMX(3-isobutyl-1-methylxanthine), paraxanthine, pentoxifylline,theobromine, theophylline, methylated xanthines, vinpocetine, EHNA(erythro-9-(2-hydroxy-3-nonyl)adenine), anagrelide, enoximone (PERFAN™),milrinone, levosimendon, mesembrine, ibudilast, piclamilast, luteolin,drotaverine, roflumilast (DAXAS™, DALIRESP™), sildenafil (REVATION®,VIAGRA®), tadalafil (ADCIRCA®, CIALIS®), vardenafil (LEVITRA®, STAXYN®),udenafil, avanafil, icariin, 4-methylpiperazine, and pyrazolopyrimidin-7-1.

Examples of proteasome inhibitors include bortezomib, disulfuram,epigallocatechin-3-gallate, and salinosporamide A.

Examples of kinase inhibitors include bevacizumab, BIBW 2992, cetuximab(ERBITUX®), imatinib (GLEEVEC®), trastuzumab (HERCEPTIN®), gefitinib(IRESSA®), ranibizumab (LUCENTIS®), pegaptanib, sorafenib, dasatinib,sunitinib, erlotinib, nilotinib, lapatinib, panitumumab, vandetanib,E7080, pazopanib, mubritinib.

Examples of glucocorticoids include hydrocortisone (cortisol), cortisoneacetate, prednisone, prednisolone, methylprednisolone, dexamethasone,betamethasone, triamcinolone, beclometasone, fludrocortisone acetate,deoxycorticosterone acetate (DOCA), and aldosterone.

Examples of retinoids include retinol, retinal, tretinoin (retinoicacid, RETIN-A®), isotretinoin (ACCUTANE®, AMNESTEEM®, CLARAVIS®,SOTRET®), alitretinoin (PANRETIN®), etretinate (TEGISON) and itsmetabolite acitretin (SORIATANE®), tazarotene (TAZORAC®, AVAGE®,ZORAC®), bexarotene (TARGRETIN®), and adapalene (DIFFERIN®).

Examples of cytokine inhibitors include IL1ra, IL1 receptor antagonist,IGFBP, TNF-BF, uromodulin, Alpha-2-Macroglobulin, Cyclosporin A,Pentamidine, and Pentoxifylline (PENTOPAK®, PENTOXIL®, TRENTAL®).

Examples of peroxisome proliferator-activated receptor antagonistsinclude GW9662, PPARγ antagonist III, G335, T0070907 (EMD4Biosciences,USA).

Examples of peroxisome proliferator-activated receptor agonists includepioglitazone, ciglitazone, clofibrate, GW1929, GW7647, L-165,041, LY171883, PPARγ activator, Fmoc-Leu, troglitazone, and WY-14643(EMD4Biosciences, USA).

Examples of histone deacetylase inhibitors include hydroxamic acids (orhydroxamates) such as trichostatin A, cyclic tetrapeptides (such astrapoxin B) and depsipeptides, benzamides, electrophilic ketones,aliphatic acid compounds such as phenylbutyrate and valproic acid,hydroxamic acids such as vorinostat (SAHA), belinostat (PXD101), LAQ824,and panobinostat (LBH589), benzamides such as entinostat (MS-275),CI994, and mocetinostat (MGCD0103), nicotinamide, derivatives of NAD,dihydrocoumarin, naphthopyranone, and 2-hydroxynaphaldehydes.

Examples of calcineurin inhibitors include cyclosporine, pimecrolimus,voclosporin, and tacrolimus.

Examples of phosphatase inhibitors include BN82002 hydrochloride,CP-91149, calyculin A, cantharidic acid, cantharidin, cypermethrin,ethyl-3,4-dephostatin, fostriecin sodium salt, MAZ51,methyl-3,4-dephostatin, NSC 95397, norcantharidin, okadaic acid ammoniumsalt from prorocentrum concavum, okadaic acid, okadaic acid potassiumsalt, okadaic acid sodium salt, phenylarsine oxide, various phosphataseinhibitor cocktails, protein phosphatase 1C, protein phosphatase 2Ainhibitor protein, protein phosphatase 2A1, protein phosphatase 2A2,sodium orthovanadate.

In some embodiments, antigens as described herein are also coupled tosynthetic nanocarriers. In some embodiments, the antigens are coupled tothe same or different synthetic nanocarriers as to which theimmunosuppressants are coupled. In other embodiments, the antigens arenot coupled to any synthetic nanocarriers. Antigens include any of theantigens provided herein, or fragments or derivatives thereof, suchantigens are associated with inflammatory, autoimmune diseases, allergy,organ or tissue rejection, graft versus host disease, transplantantigens and therapeutic protein antigens. The epitopes, or proteins,polypeptides or peptides that comprise the epitopes, can be obtained orderived from any of the antigens provided or otherwise known in the art.

Therapeutic proteins include, but are not limited to, infusibletherapeutic proteins, enzymes, enzyme cofactors, hormones, bloodclotting factors, cytokines and interferons, growth factors, monoclonalantibodies, and polyclonal antibodies (e.g., that are administered to asubject as a replacement therapy), and proteins associated with Pompe'sdisease (e.g., alglucosidase alfa, rhGAA (e.g., Myozyme and Lumizyme(Genzyme)). Therapeutic proteins also include proteins involved in theblood coagulation cascade. Therapeutic proteins include, but are notlimited to, Factor VIII, Factor VII, Factor IX, Factor V, von WillebrandFactor, von Heldebrant Factor, tissue plasminogen activator, insulin,growth hormone, erythropoietin alfa, VEGF, thrombopoietin, lysozyme,antithrombin and the like. Therapeutic proteins also include adipokines,such as leptin and adiponectin. Other examples of therapeutic proteinsare as described below and elsewhere herein. Also included are fragmentsor derivatives of any of the therapeutic proteins provided as theantigen.

Examples of therapeutic proteins used in enzyme replacement therapy ofsubjects having a lysosomal storage disorder include, but are notlimited to, imiglucerase for the treatment of Gaucher's disease (e.g.,CEREZYME™), a-galactosidase A (a-gal A) for the treatment of Fabrydisease (e.g., agalsidase beta, FABRYZYME™), acid α-glucosidase (GAA)for the treatment of Pompe disease (e.g., alglucosidase alfa, LUMIZYME,MYOZYME), arylsulfatase B for the treatment of Mucopolysaccharidoses(e.g., laronidase, ALDURAZYME™, idursulfase, ELAPRASE™, arylsulfatase B,NAGLAZYME™).

Examples of enzymes include oxidoreductases, transferases, hydrolases,lyases, isomerases, and ligases.

Examples of hormones include Melatonin (N-acetyl-5-methoxytryptamine),Serotonin, Thyroxine (or tetraiodothyronine) (a thyroid hormone),Triiodothyronine (a thyroid hormone), Epinephrine (or adrenaline),Norepinephrine (or noradrenaline), Dopamine (or prolactin inhibitinghormone), Antimullerian hormone (or mullerian inhibiting factor orhormone), Adiponectin, Adrenocorticotropic hormone (or corticotropin),Angiotensinogen and angiotensin, Antidiuretic hormone (or vasopressin,arginine vasopressin), Atrial-natriuretic peptide (or atriopeptin),Calcitonin, Cholecystokinin, Corticotropin-releasing hormone,Erythropoietin, Follicle-stimulating hormone, Gastrin, Ghrelin,Glucagon, Glucagon-like peptide (GLP-1), GIP, Gonadotropin-releasinghormone, Growth hormone-releasing hormone, Human chorionic gonadotropin,Human placental lactogen, Growth hormone, Inhibin, Insulin, Insulin-likegrowth factor (or somatomedin), Leptin, Luteinizing hormone, Melanocytestimulating hormone, Orexin, Oxytocin, Parathyroid hormone, Prolactin,Relaxin, Secretin, Somatostatin, Thrombopoietin, Thyroid-stimulatinghormone (or thyrotropin), Thyrotropin-releasing hormone, Cortisol,Aldosterone, Testosterone, Dehydroepiandrosterone, Androstenedione,Dihydrotestosterone, Estradiol, Estrone, Estriol, Progesterone,Calcitriol (1,25-dihydroxyvitamin D3), Calcidiol (25-hydroxyvitamin D3),Prostaglandins, Leukotrienes, Prostacyclin, Thromboxane, Prolactinreleasing hormone, Lipotropin, Brain natriuretic peptide, NeuropeptideY, Histamine, Endothelin, Pancreatic polypeptide, Renin, and Enkephalin.

Examples of blood and blood coagulation factors include Factor I(fibrinogen), Factor II (prothrombin), tissue factor, Factor V(proaccelerin, labile factor), Factor VII (stable factor, proconvertin),Factor VIII (antihemophilic globulin), Factor IX (Christmas factor orplasma thromboplastin component), Factor X (Stuart-Prower factor),Factor Xa, Factor XI, Factor XII (Hageman factor), Factor XIII(fibrin-stabilizing factor), von Willebrand factor, prekallikrein(Fletcher factor), high-molecular weight kininogen (HMWK) (Fitzgeraldfactor), fibronectin, fibrin, thrombin, antithrombin III, heparincofactor II, protein C, protein S, protein Z, protein Z-related proteaseinhibitot (ZPI), plasminogen, alpha 2-antiplasmin, tissue plasminogenactivator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI1),plasminogen activator inhibitor-2 (PAI2), cancer procoagulant, andepoetin alfa (Epogen, Procrit).

Examples of cytokines include lymphokines, interleukins, and chemokines,type 1 cytokines, such as IFN-γ, TGF-β, and type 2 cytokines, such asIL-4, IL-10, and IL-13.

Examples of growth factors include Adrenomedullin (AM), Angiopoietin(Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs),Brain-derived neurotrophic factor (BDNF), Epidermal growth factor (EGF),Erythropoietin (EPO), Fibroblast growth factor (FGF), Glial cellline-derived neurotrophic factor (GDNF), Granulocyte colony-stimulatingfactor (G-CSF), Granulocyte macrophage colony-stimulating factor(GM-CSF), Growth differentiation factor-9 (GDF9), Hepatocyte growthfactor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growthfactor (IGF), Migration-stimulating factor, Myostatin (GDF-8), Nervegrowth factor (NGF) and other neurotrophins, Platelet-derived growthfactor (PDGF), Thrombopoietin (TPO), Transforming growth factor alpha(TGF-α), Transforming growth factor beta (TGF-β),Tumour_necrosis_factor-alpha (TNF-α), Vascular endothelial growth factor(VEGF), Wnt Signaling Pathway, placental growth factor (P1GF), [(FoetalBovine Somatotrophin)] (FBS), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, andIL-7.

Examples of monoclonal antibodies include Abagovomab, Abciximab,Adalimumab, Adecatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD,Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, Anrukinzumab,Anti-thymocyte globin, Apolizumab, Arcitumomab, Aselizumab, Atlizumab(tocilizumab), Atorolimumab, Bapineuzumab, Basiliximab, Bavituximab,Bectumomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab,Bevacizumab, Biciromab, Bivatuzumab mertansine, Blinatumomab,Brentuximab vedotin, Briakinumab, Canakinumab, Cantuzumab mertansine,Capromab pendetide, Catumaxomab, Cedelizumab, Certolizumab pegol,Cetuximab, Citatuzumab bogatox, Cixutumumab, Clenoliximab, Clivatuzumabtetraxetan, Conatumumab, Dacetuzumab, Daclizumab, Daratumumab,Denosumab, Detumomab, Dorlimomab aritox, Dorlixizumab, Ecromeximab,Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Elotuzumab,Elsilimomab, Enlimomab pegol, Epitumomab cituxetan, Epratuzumab,Erlizumab, Ertumaxomab, Etaracizumab, Exbivirumab, Fanolesomab,Faralimomab, Farletuzumab, Felvizumab, Fezakinumab, Figitumumab,Fontolizumab, Foravirumab, Fresolimumab, Galiximab, Gantenerumab,Gavilimomab, Gemtuzumab ozogamicin, GC1008, Girentuximab, Glembatumumabvedotin, Golimumab, Gomiliximab, Ibalizumab, Ibritumomab tiuxetan,Igovomab, Imciromab, Infliximab, Intetumumab, Inolimomab, Inotuzumabozogamicin, Ipilimumab, Iratumumab, Keliximab, Labetuzumab,Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab,Lintuzumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab,Mapatumumab, Maslimomab, Matuzumab, Mepolizumab, Metelimumab,Milatuzumab, Minretumomab, Mitumomab, Morolimumab, Motavizumab,Muromonab-CD3, Nacolomab tafenatox, Naptumomab estafenatox, Natalizumab,Nebacumab, Necitumumab, Nerelimomab, Nimotuzumab, Nofetumomab merpentan,Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Omalizumab, Oportuzumabmonatox, Oregovomab, Otelixizumab, Pagibaximab, Palivizumab,Panitumumab, Panobacumab, Pascolizumab, Pemtumomab, Pertuzumab,Pexelizumab, Pintumomab, Priliximab, Pritumumab, Rafivirumab,Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab Reslizumab,Rilotumumab, Rituximab, Robatumumab, Rontalizumab, Rovelizumab,Ruplizumab, Satumomab pendetide, Sevirumab, Sibrotuzumab, Sifalimumab,Siltuximab, Siplizumab, Solanezumab, Sonepcizumab, Sontuzumab,Stamulumab, Sulesomab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab,Tanezumab, Taplitumomab paptox, Tefibazumab, Telimomab aritox,Tenatumomab, Teneliximab, Teplizumab, Ticilimumab (tremelimumab),Tigatuzumab, Tocilizumab (atlizumab), Toralizumab, Tositumomab,Trastuzumab, Tremelimumab, Tucotuzumab celmoleukin, Tuvirumab,Urtoxazumab, Ustekinumab, Vapaliximab, Vedolizumab, Veltuzumab,Vepalimomab, Visilizumab, Volociximab, Votumumab, Zalutumumab,Zanolimumab, Ziralimumab, and Zolimomab aritox.

Examples of infusion therapy or injectable therapeutic proteins include,for example, Tocilizumab (Roche/Actemra®), alpha-1 antitrypsin(Kamada/AAT), Hematide® (Affymax and Takeda, synthetic peptide),albinterferon alfa-2b (Novartis/Zalbin™), Rhucin® (Pharming Group, C1inhibitor replacement therapy), tesamorelin (Theratechnologies/Egrifta,synthetic growth hormone-releasing factor), ocrelizumab (Genentech,Roche and Biogen), belimumab (GlaxoSmithKline/Benlysta®), pegloticase(Savient Pharmaceuticals/Krystexxa™), taliglucerase alfa(Protalix/Uplyso), agalsidase alfa (Shire/Replagal®), velaglucerase alfa(Shire).

Additional therapeutic proteins useful in accordance to aspects of thisinvention will be apparent to those of skill in the art, and theinvention is not limited in this respect.

In some embodiments, a component, such as an antigen orimmunosuppressant, may be isolated. Isolated refers to the element beingseparated from its native environment and present in sufficientquantities to permit its identification or use. This means, for example,the element may be (i) selectively produced by expression cloning or(ii) purified as by chromatography or electrophoresis. Isolated elementsmay be, but need not be, substantially pure. Because an isolated elementmay be admixed with a pharmaceutically acceptable excipient in apharmaceutical preparation, the element may comprise only a smallpercentage by weight of the preparation. The element is nonethelessisolated in that it has been separated from the substances with which itmay be associated in living systems, i.e., isolated from other lipids orproteins. Any of the elements provided herein can be included in thecompositions in isolated form.

D. Methods of Making and Using the Inventive Compositions and RelatedMethods

Synthetic nanocarriers may be prepared using a wide variety of methodsknown in the art. For example, synthetic nanocarriers can be formed bymethods as nanoprecipitation, flow focusing fluidic channels, spraydrying, single and double emulsion solvent evaporation, solventextraction, phase separation, milling, microemulsion procedures,microfabrication, nanofabrication, sacrificial layers, simple andcomplex coacervation, and other methods well known to those of ordinaryskill in the art. Alternatively or additionally, aqueous and organicsolvent syntheses for monodisperse semiconductor, conductive, magnetic,organic, and other nanomaterials have been described (Pellegrino et al.,2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; andTrindade et al., 2001, Chem. Mat., 13:3843). Additional methods havebeen described in the literature (see, e.g., Doubrow, Ed.,“Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press,Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5:13;Mathiowitz et al., 1987, Reactive Polymers, 6:275; and Mathiowitz etal., 1988, J. Appl. Polymer Sci., 35:755; U.S. Pat. Nos. 5,578,325 and6,007,845; P. Paolicelli et al., “Surface-modified PLGA-basedNanoparticles that can Efficiently Associate and Deliver Virus-likeParticles” Nanomedicine. 5(6):843-853 (2010)).

Various materials may be encapsulated into synthetic nanocarriers asdesirable using a variety of methods including but not limited to C.Astete et al., “Synthesis and characterization of PLGA nanoparticles” J.Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp. 247-289 (2006); K.Avgoustakis “Pegylated Poly(Lactide) and Poly(Lactide-Co-Glycolide)Nanoparticles: Preparation, Properties and Possible Applications in DrugDelivery” Current Drug Delivery 1:321-333 (2004); C. Reis et al.,“Nanoencapsulation I. Methods for preparation of drug-loaded polymericnanoparticles” Nanomedicine 2:8-21 (2006); P. Paolicelli et al.,“Surface-modified PLGA-based Nanoparticles that can EfficientlyAssociate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853(2010). Other methods suitable for encapsulating materials intosynthetic nanocarriers may be used, including without limitation methodsdisclosed in U.S. Pat. No. 6,632,671 to Unger Oct. 14, 2003.

In certain embodiments, synthetic nanocarriers are prepared by ananoprecipitation process or spray drying. Conditions used in preparingsynthetic nanocarriers may be altered to yield particles of a desiredsize or property (e.g., hydrophobicity, hydrophilicity, externalmorphology, “stickiness,” shape, etc.). The method of preparing thesynthetic nanocarriers and the conditions (e.g., solvent, temperature,concentration, air flow rate, etc.) used may depend on the materials tobe coupled to the synthetic nanocarriers and/or the composition of thepolymer matrix.

If particles prepared by any of the above methods have a size rangeoutside of the desired range, particles can be sized, for example, usinga sieve.

Elements (i.e., components) of the inventive synthetic nanocarriers(such as moieties of which an immunofeature surface is comprised,targeting moieties, polymeric matrices, antigens, immunosuppressants andthe like) may be coupled to the overall synthetic nanocarrier, e.g., byone or more covalent bonds, or may be coupled by means of one or morelinkers. Additional methods of functionalizing synthetic nanocarriersmay be adapted from Published US Patent Application 2006/0002852 toSaltzman et al., Published US Patent Application 2009/0028910 toDeSimone et al., or Published International Patent ApplicationWO/2008/127532 A1 to Murthy et al.

Alternatively or additionally, synthetic nanocarriers can be coupled tocomponents directly or indirectly via non-covalent interactions. Innon-covalent embodiments, the non-covalent coupling is mediated bynon-covalent interactions including but not limited to chargeinteractions, affinity interactions, metal coordination, physicaladsorption, host-guest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof. Such couplingsmay be arranged to be on an external surface or an internal surface ofan inventive synthetic nanocarrier. In embodiments, encapsulation and/orabsorption is a form of coupling. In embodiments, the inventivesynthetic nanocarriers can be combined with an antigen by admixing inthe same vehicle or delivery system.

Populations of synthetic nanocarriers may be combined to formpharmaceutical dosage forms according to the present invention usingtraditional pharmaceutical mixing methods. These include liquid-liquidmixing in which two or more suspensions, each containing one or moresubsets of nanocarriers, are directly combined or are brought togethervia one or more vessels containing diluent. As synthetic nanocarriersmay also be produced or stored in a powder form, dry powder-powdermixing could be performed as could the re-suspension of two or morepowders in a common media. Depending on the properties of thenanocarriers and their interaction potentials, there may be advantagesconferred to one or another route of mixing.

Typical inventive compositions that comprise synthetic nanocarriers maycomprise inorganic or organic buffers (e.g., sodium or potassium saltsof phosphate, carbonate, acetate, or citrate) and pH adjustment agents(e.g., hydrochloric acid, sodium or potassium hydroxide, salts ofcitrate or acetate, amino acids and their salts) antioxidants (e.g.,ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20,polysorbate 80, polyoxyethylene9-10 nonyl phenol, sodium desoxycholate),solution and/or cryo/lyo stabilizers (e.g., sucrose, lactose, mannitol,trehalose), osmotic adjustment agents (e.g., salts or sugars),antibacterial agents (e.g., benzoic acid, phenol, gentamicin),antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g.,thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers andviscosity-adjustment agents (e.g., polyvinylpyrrolidone, poloxamer 488,carboxymethylcellulose) and co-solvents (e.g., glycerol, polyethyleneglycol, ethanol).

Compositions according to the invention comprise inventive syntheticnanocarriers in combination with pharmaceutically acceptable excipients.The compositions may be made using conventional pharmaceuticalmanufacturing and compounding techniques to arrive at useful dosageforms. Techniques suitable for use in practicing the present inventionmay be found in Handbook of Industrial Mixing: Science and Practice,Edited by Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta,2004 John Wiley & Sons, Inc.; and Pharmaceutics: The Science of DosageForm Design, 2nd Ed. Edited by M. E. Auten, 2001, Churchill Livingstone.In an embodiment, inventive synthetic nanocarriers are suspended insterile saline solution for injection together with a preservative.

It is to be understood that the compositions of the invention can bemade in any suitable manner, and the invention is in no way limited tocompositions that can be produced using the methods described herein.Selection of an appropriate method may require attention to theproperties of the particular moieties being associated.

In some embodiments, inventive synthetic nanocarriers are manufacturedunder sterile conditions or are terminally sterilized. This can ensurethat resulting compositions are sterile and non-infectious, thusimproving safety when compared to non-sterile compositions. Thisprovides a valuable safety measure, especially when subjects receivingsynthetic nanocarriers have immune defects, are suffering frominfection, and/or are susceptible to infection. In some embodiments,inventive synthetic nanocarriers may be lyophilized and stored insuspension or as lyophilized powder depending on the formulationstrategy for extended periods without losing activity.

The compositions of the invention can be administered by a variety ofroutes, including but not limited to subcutaneous, intranasal, oral,intravenous, intraperitoneal, intramuscular, transmucosal, transmucosal,sublingual, rectal, ophthalmic, pulmonary, intradermal, transdermal,transcutaneous or intradermal or by a combination of these routes.Routes of administration also include administration by inhalation orpulmonary aerosol. Techniques for preparing aerosol delivery systems arewell known to those of skill in the art (see, for example, Sciarra andCutie, “Aerosols,” in Remington's Pharmaceutical Sciences, 18th edition,1990, pp. 1694-1712; incorporated by reference).

The transplantable grafts or therapeutic proteins provided as acell-based therapy of the invention may be administered by parenteral,intraarterial, intranasal or intravenous administration or by injectionto lymph nodes or anterior chamber of the eye or by local administrationto an organ or tissue of interest. The administration may be bysubcutaneous, intrathecal, intraventricular, intramuscular,intraperitoneal, intracoronary, intrapancreatic, intrahepatic orbronchial injection.

The compositions of the invention can be administered in effectiveamounts, such as the effective amounts described elsewhere herein. Dosesof dosage forms contain varying amounts of populations of syntheticnanocarriers and/or varying amounts of immunosuppressants and/orantigens, according to the invention. The amount of syntheticnanocarriers and/or immunosuppressants and/or antigens present in theinventive dosage forms can be varied according to the nature of theantigens, the therapeutic benefit to be accomplished, and other suchparameters. In embodiments, dose ranging studies can be conducted toestablish optimal therapeutic amount of the population of syntheticnanocarriers and the amount of immunosuppressants and/or antigens to bepresent in the dosage form. In embodiments, the synthetic nanocarriersand/or the immunosuppressants and/or antigens are present in the dosageform in an amount effective to generate a tolerogenic immune response tothe antigens upon administration to a subject. It may be possible todetermine amounts of the immunosuppressants and/or antigens effective togenerate a tolerogenic immune response using conventional dose rangingstudies and techniques in subjects. Inventive dosage forms may beadministered at a variety of frequencies. In a preferred embodiment, atleast one administration of the dosage form is sufficient to generate apharmacologically relevant response. In more preferred embodiments, atleast two administrations, at least three administrations, or at leastfour administrations, of the dosage form are utilized to ensure apharmacologically relevant response.

Prophylactic administration of the inventive compositions can beinitiated prior to the onset of disease, disorder or condition ortherapeutic administration can be initiated after a disorder, disorderor condition is established.

In some embodiments, administration of synthetic nanocarriers isundertaken e.g., prior to administration of a therapeutic protein ortransplantable graft or exposure to an allergen. In exemplaryembodiments, synthetic nanocarriers are administered at one or moretimes including, but not limited to, 30, 25, 20, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 days prior to administration of atherapeutic protein or transplantable graft or exposure to an allergen.In addition or alternatively, synthetic nanocarriers can be administeredto a subject following administration of a therapeutic protein ortransplantable graft or exposure to an allergen. In exemplaryembodiments, synthetic nanocarriers are administered at one or moretimes including, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 20, 25, 30, etc. days following administration of atherapeutic protein or transplantable graft or exposure to an allergen.

In some embodiments, a maintenance dose (e.g., of a syntheticnanocarrier composition provided herein) is administered to a subjectafter an initial administration has resulted in a tolerogenic responsein the subject, for example to maintain the tolerogenic effect achievedafter the initial dose, to prevent an undesired immune reaction in thesubject, or to prevent the subject becoming a subject at risk ofexperiencing an undesired immune response or an undesired level of animmune response. In some embodiments, the maintenance dose is the samedose as the initial dose the subject received. In some embodiments, themaintenance dose is a lower dose than the initial dose. For example, insome embodiments, the maintenance dose is about 3/4, about 2/3, about1/2, about 1/3, about 1/4, about 1/8, about 1/10, about 1/20, about1/25, about 1/50, about 1/100, about 1/1,000, about 1/10,000, about1/100,000, or about 1/1,000,000 (weight/weight) of the initial dose.

The compositions and methods described herein can be used to induce orenhance a tolerogenic immune response and/or to suppress, modulate,direct or redirect an undesired immune response for the purpose ofimmune suppression. The compositions and methods described herein can beused in the diagnosis, prophylaxis and/or treatment of diseases,disorders or conditions in which immune suppression (e.g., tolerogenicimmune response) would confer a treatment benefit. Such diseases,disorders or conditions include inflammatory diseases, autoimmunediseases, allergies, organ or tissue rejection and graft versus hostdisease. The compositions and methods described herein can also be usedin subjects who have undergone or will undergo transplantation. Thecompositions and methods described herein can also be used in subjectswho have received, are receiving or will receive a therapeutic proteinagainst which they have generated or are expected to generate anundesired immune response.

Autoimmune diseases include, but are not limited to, rheumatoidarthritis, multiple sclerosis, immune-mediated or Type I diabetesmellitus, inflammatory bowel disease (e.g., Crohn's disease orulcerative colitis), systemic lupus erythematosus, psoriasis,scleroderma, autoimmune thyroid disease, alopecia greata, Grave'sdisease, Guillain-Barré syndrome, celiac disease, Sjögren's syndrome,rheumatic fever, gastritis, autoimmune atrophic gastritis, autoimmunehepatitis, insulitis, oophoritis, orchitis, uveitis, phacogenic uveitis,myasthenia gravis, primary myxoedema, pernicious anemia, autoimmunehaemolytic anemia, Addison's disease, scleroderma, Goodpasture'ssyndrome, nephritis, for example, glomerulonephritis, psoriasis,pemphigus vulgaris, pemphigoid, sympathetic opthalmia, idiopathicthrombocylopenic purpura, idiopathic feucopenia, Wegener'sgranulomatosis and poly/dermatomyositis.

Some additional exemplary autoimmune diseases, associated autoantigens,and autoantibodies, which are contemplated for use in the invention, aredescribed in Table 1 below:

Autoantibody Type Autoantibody Autoantigen Autoimmune disease ordisorder Antinuclear Anti-SSA/Ro ribonucleoproteins Systemic lupuserythematosus, neonatal antibodies autoantibodies heart block, primarySjögren's syndrome Anti-La/SS-B ribonucleoproteins Primary Sjögren'ssyndrome autoantibodies Anti-centromere centromere CREST syndromeantibodies Anti-neuronal Ri[disambiguation Opsoclonus nuclear antibody-2needed] Anti-dsDNA double-stranded SLE DNA Anti-Jo1 histidine-tRNAInflammatory myopathy ligase Anti-Smith snRNP core proteins SLE Anti-Type I Systemic sclerosis (anti-Scl-70 antibodies) topoisomerasetopoisomerase antibodies Anti-histone histones SLE and Drug-inducedLE[2] antibodies Anti-p62 nucleoporin 62 Primary biliarycirrhosis[3][4][5] antibodies[3] Anti-sp100 Sp100 nuclear antibodies [4]antigen Anti-glycoprotein- nucleoporin 210 kDa 210 antibodies[5] Anti-Anti-tTG Coeliac disease transglutaminase Anti-eTG Dermatitisherpetiformis antibodies Anti-ganglioside ganglioside GQ1B Miller-FisherSyndrome antibodies ganglioside GD3 Acute motor axonal neuropathy (AMAN)ganglioside GM1 Multifocal motor neuropathy with conduction block (MMN)Anti-actin actin Coeliac disease anti-actin antibodies antibodiescorrelated with the level of intestinal damage [6][7] Liver kidneyAutoimmune hepatitis.[8] microsomal type 1 antibody Lupus anticoagulantAnti-thrombin thrombin Systemic lupus erythematosus antibodiesAnti-neutrophil phospholipid Antiphospholipid syndrome cytoplasmicc-ANCA proteins in Wegener's granulomatosis antibody neutrophilcytoplasm p-ANCA neutrophil Microscopic polyangiitis, Churg-Straussperinuclear syndrome, systemic vasculitides (non- specific) Rheumatoidfactor IgG Rheumatoid arthritis Anti-smooth muscle smooth muscle Chronicautoimmune hepatitis antibody Anti-mitochondrial mitochondria Primarybiliary cirrhosis[9] antibody Anti-SRP signal recognitionPolymyositis[10] particle exosome complex Scleromyositis nicotinicMyasthenia gravis acetylcholine receptor muscle-specific Myastheniagravis kinase (MUSK) Anti-VGCC voltage-gated Lambert-Eaton myasthenicsyndrome calcium channel (P/Q-type) thyroid peroxidase Hashimoto'sthyroiditis (microsomal) TSH receptor Graves' disease Hu Paraneoplasticcerebellar syndrome Yo (cerebellar Paraneoplastic cerebellar syndromePurkinje Cells) amphiphysin Stiff person syndrome, paraneoplasticcerebellar syndrome Anti-VGKC voltage-gated Limbic encephalitis, Isaac'sSyndrome potassium channel (autoimmune neuromyotonia) (VGKC) basalganglia Sydenham's chorea, paediatric autoimmune neuronsneuropsychiatric disease associated with Streptococcus (PANDAS)N-methyl-D- Encephalitis aspartate receptor (NMDA) glutamic acidDiabetes mellitus type 1, stiff person decarboxylase syndrome (GAD)aquaporin-4 Neuromyelitis optica (Devic's syndrome)

Inflammatory diseases include, but are not limited to, Alzheimer's,Ankylosing spondylitis, arthritis, asthma, atherosclerosis, Behcet'sdisease, chronic inflammatory demyelinating polyradiculoneuropathy,Crohn's disease, colitis, cystic fibrosis, dermatitis, diverticulitis,hepatitis, irritable bowel syndrome (IBS), lupus erythematous, musculardystrophy, nephritis, Parkinson's, shingles and ulcerative colitis.Inflammatory diseases also include, for example, cardiovascular disease,chronic obstructive pulmonary disease (COPD), bronchiectasis, chroniccholecystitis, tuberculosis, Hashimoto's thyroiditis, sepsis,sarcoidosis, silicosis and other pneumoconioses, and an implantedforeign body in a wound, but are not so limited. As used herein, theterm “sepsis” refers to a well-recognized clinical syndrome associatedwith a host's systemic inflammatory response to microbial invasion. Theterm “sepsis” as used herein refers to a condition that is typicallysignaled by fever or hypothermia, tachycardia, and tachypnea, and insevere instances can progress to hypotension, organ dysfunction, andeven death.

In some embodiments, the inflammatory disease is non-autoimmuneinflammatory bowel disease, post-surgical adhesions, coronary arterydisease, hepatic fibrosis, acute respiratory distress syndrome, acuteinflammatory pancreatitis, endoscopic retrogradecholangiopancreatography-induced pancreatitis, burns, atherogenesis ofcoronary, cerebral and peripheral arteries, appendicitis, cholecystitis,diverticulitis, visceral fibrotic disorders, wound healing, skinscarring disorders (keloids, hidradenitis suppurativa), granulomatousdisorders (sarcoidosis, primary biliary cirrhosis), asthma, pyodermagandrenosum, Sweet's syndrome, Behcet's disease, primary sclerosingcholangitis or an abscess. In some preferred embodiment the inflammatorydisease is inflammatory bowel disease (e.g., Crohn's disease orulcerative colitis).

In other embodiments, the inflammatory disease is an autoimmune disease.The autoimmune disease in some embodiments is rheumatoid arthritis,rheumatic fever, ulcerative colitis, Crohn's disease, autoimmuneinflammatory bowel disease, insulin-dependent diabetes mellitus,diabetes mellitus, juvenile diabetes, spontaneous autoimmune diabetes,gastritis, autoimmune atrophic gastritis, autoimmune hepatitis,thyroiditis, Hashimoto's thyroiditis, insulitis, oophoritis, orchitis,uveitis, phacogenic uveitis, multiple sclerosis, myasthenia gravis,primary myxoedema, thyrotoxicosis, pernicious anemia, autoimmunehaemolytic anemia, Addison's disease, Anklosing spondylitis,sarcoidosis, scleroderma, Goodpasture's syndrome, Guillain-Barrésyndrome, Graves' disease, glomerulonephritis, psoriasis, pemphigusvulgaris, pemphigoid, excema, bulous pemiphigous, sympathetic opthalmia,idiopathic thrombocylopenic purpura, idiopathic feucopenia, Sjogren'ssyndrome, systemic sclerosis, Wegener's granulomatosis,poly/dermatomyositis, primary biliary cirrhosis, primary sclerosingcholangitis, lupus or systemic lupus erythematosus.

Graft versus host disease (GVHD) is a complication that can occur aftera pluripotent cell (e.g., stem cell) or bone marrow transplant in whichthe newly transplanted material results in an attack on the transplantrecipient's body. In some instances, GVHD takes place after a bloodtransfusion. Graft-versus-host-disease can be divided into acute andchronic forms. The acute or fulminant form of the disease (aGVHD) isnormally observed within the first 100 days post-transplant, and is amajor challenge to transplants owing to associated morbidity andmortality. The chronic form of graft-versus-host-disease (cGVHD)normally occurs after 100 days. The appearance of moderate to severecases of cGVHD adversely influences long-term survival.

EXAMPLES Example 1 Immune Response of Synthetic Nanocarriers withCoupled Rapamycin with and without Ovalbumin Peptide (323-339)

Materials

Ovalbumin peptide 323-339, a 17 amino acid peptide known to be a T and Bcell epitope of Ovalbumin protein, was purchased from Bachem AmericasInc. (3132 Kashiwa Street, Torrance Calif. 90505; Part #4065609).Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham,Mass. 01702; Product Catalogue #R1017). PLGA with a lactide:glycolideratio of 3:1 and an inherent viscosity of 0.75 dL/g was purchased fromSurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211;Product Code 7525 DLG 7A). Polyvinyl alcohol (85-89% hydrolyzed) waspurchased from EMD Chemicals (Product Number 1.41350.1001).

Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute hydrochloricacid aqueous solution. The solution was prepared by dissolving ovalbuminpeptide in 0.13 M hydrochloric acid solution at room temperature.

Solution 2: Rapamycin @ 50 mg/mL in methylene chloride. The solution wasprepared by dissolving rapamycin in pure methylene chloride.

Solution 3: PLGA @ 100 mg/mL in methylene chloride. The solution wasprepared by dissolving PLGA in pure methylene chloride.

Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphatebuffer.

Method for Preparing Synthetic Nanocarrier Containing Rapamycin andOvalbumin (323-339)

A primary water-in-oil emulsion was prepared first. W1/O1 was preparedby combining solution 1 (0.2 mL), solution 2 (0.2 mL), and solution 3(1.0 mL) in a small pressure tube and sonicating at 50% amplitude for 40seconds using a Branson Digital Sonifier 250. A secondary emulsion(W1/O1/W2) was then prepared by combining solution 4 (3.0 mL) with theprimary W1/O1 emulsion, vortexing for 10 s, and sonicating at 30%amplitude for 60 seconds using the Branson Digital Sonifier 250.

The W1/O1/W2 emulsion was added to a beaker containing 70 mM pH 8phosphate buffer solution (30 mL) and stirred at room temperature for 2hours to allow the methylene chloride to evaporate and for the syntheticnanocarriers to form. A portion of the synthetic nanocarriers werewashed by transferring the synthetic nanocarrier suspension to acentrifuge tube and centrifuging at 21,000×g and 4° C. for one hour,removing the supernatant, and re-suspending the pellet in phosphatebuffered saline. The washing procedure was repeated, and the pellet wasre-suspended in phosphate buffered saline for a final syntheticnanocarrier dispersion of about 10 mg/mL.

The amounts of peptide and rapamycin in the synthetic nanocarriers weredetermined by HPLC analysis. The total dry-synthetic nanocarrier massper mL of suspension was determined by a gravimetric method.

Method for Synthetic Nanocarrier Containing Rapamycin

A primary water-in-oil emulsion was prepared first. W1/O1 was preparedby combining 0.13 M hydrochloric acid solution (0.2 mL), solution 2 (0.2mL), and solution 3 (1.0 mL) in a small pressure tube and sonicating at50% amplitude for 40 seconds using a Branson Digital Sonifier 250. Asecondary emulsion (W1/O1/W2) was then prepared by combining solution 4(3.0 mL) with the primary W1/O1 emulsion, vortexing for 10 s, andsonicating at 30% amplitude for 60 seconds using the Branson DigitalSonifier 250.

The W1/O1/W2 emulsion was added to a beaker containing 70 mM pH 8phosphate buffer solution (30 mL) and stirred at room temperature for 2hours to allow the methylene chloride to evaporate and for the syntheticnanocarriers to form. A portion of the synthetic nanocarriers werewashed by transferring the synthetic nanocarrier suspension to acentrifuge tube and centrifuging at 21,000×g and 4° C. for one hour,removing the supernatant, and re-suspending the pellet in phosphatebuffered saline. The washing procedure was repeated, and the pellet wasre-suspended in phosphate buffered saline for a final syntheticnanocarrier dispersion of about 10 mg/mL.

The amount of rapamycin in the synthetic nanocarrier was determined byHPLC analysis. The total dry-synthetic nanocarrier mass per mL ofsuspension was determined by a gravimetric method.

Method for Measuring Rapamycin Load

Approximately 3 mg of synthetic nanocarriers were collected andcentrifuged to separate supernatant from synthetic nanocarrier pellet.Acetonitrile was added to the pellet, and the sample was sonicated andcentrifuged to remove any insoluble material. The supernatant and pelletwere injected on RP-HPLC and absorbance was read at 278 nm. The μg foundin the pellet were used to calculate % entrapped (load), μg insupernatant and pellet were used to calculate total μg recovered.

Method for Measuring Ovalbumin (323-339) Load

Approximately 3 mg of synthetic nanocarriers were collected andcentrifuged to separate supernatant from synthetic nanocarrier pellet.Trifluoroethanol was added to the pellet and the sample was sonicated todissolve the polymer, 0.2% trifluoroacetic acid was added and sample wassonicated and then centrifuged to remove any insoluble material. Thesupernatant and pellet were injected on RP-HPLC and absorbance was readat 215 nm. The μg found in the pellet were used to calculate % entrapped(load), μg in supernatant and pellet were used to calculate total μgrecovered.

Antigen-Specific Tolerogenic Dendritic Cells (Tdc) Activity on Treg CellDevelopment

The assay included the use of OTII mice which have a transgenic T-cellreceptor specific for an immune-dominant ovalbumin (323-339). In orderto create antigen-specific tDCs, CD11c+ splenocytes were isolated, andthe ovalbumin (323-339) peptide added in vitro at 1 μg/ml or no antigen.Soluble or nanocarrier-encapsulated rapamycin was then added to the DCsfor 2 hours which were then washed extensively to remove free rapamycinfrom the culture. Purified responder CD4+CD25− cells were isolated fromOTII mice and added to tDC at a 10:1 T to DC ratio. The mixture of tDCand OTII T-cells were then cultured for 4-5 days, and the frequency ofTreg cells (CD4+CD25highFoxP3+) were analyzed by flow cytometry as shownin FIG. 1. Regions were selected based on isotype controls.

Example 2 Mesoporous Silica Nanoparticles with Coupled Ibuprofen(Prophetic)

Mesoporous SiO2 nanoparticle cores are created through a sol-gelprocess. Hexadecyltrimethyl-ammonium bromide (CTAB) (0.5 g) is dissolvedin deionized water (500 mL), and then 2 M aqueous NaOH solution (3.5 mL)is added to the CTAB solution. The solution is stirred for 30 min, andthen Tetraethoxysilane (TEOS) (2.5 mL) is added to the solution. Theresulting gel is stirred for 3 h at a temperature of 80° C. The whiteprecipitate which forms is captured by filtration, followed by washingwith deionized water and drying at room temperature. The remainingsurfactant is then extracted from the particles by suspension in anethanolic solution of HCl overnight. The particles are washed withethanol, centrifuged, and redispersed under ultrasonication. This washprocedure is repeated two additional times.

The SiO2 nanoparticles are then functionalized with amino groups using(3-aminopropyl)-triethoxysilane (APTMS). To do this, the particles aresuspended in ethanol (30 mL), and APTMS (50 μL) is added to thesuspension. The suspension is allowed to stand at room temperature for 2h and then is boiled for 4 h, keeping the volume constant byperiodically adding ethanol. Remaining reactants are removed by fivecycles of washing by centrifugation and redispersing in pure ethanol.

In a separate reaction, 1-4 nm diameter gold seeds are created. Allwater used in this reaction is first deionized and then distilled fromglass. Water (45.5 mL) is added to a 100 mL round-bottom flask. Whilestirring, 0.2 M aqueous NaOH (1.5 mL) is added, followed by a 1% aqueoussolution of tetrakis(hydroxymethyl)phosphonium chloride (THPC) (1.0 mL).Two minutes after the addition of THPC solution, a 10 mg/mL aqueoussolution of chloroauric acid (2 mL), which has been aged at least 15min, is added. The gold seeds are purified through dialysis againstwater.

To form the core-shell nanocarriers, the amino-functionalized SiO2nanoparticles formed above are first mixed with the gold seeds for 2 hat room temperature. The gold-decorated SiO2 particles are collectedthrough centrifugation and mixed with an aqueous solution of chloroauricacid and potassium bicarbonate to form the gold shell. The particles arethen washed by centrifugation and redispersed in water. Ibuprofen isloaded by suspending the particles in a solution of sodium ibuprofen (1mg/L) for 72 h. Free ibuprofen is then washed from the particles bycentrifugation and redispersing in water.

Example 3 Liposomes Containing Cyclosporine A (Prophetic)

The liposomes are formed using thin film hydration.1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (32 μmol),cholesterol (32 μmol), and cyclosporin A (6.4 μmol) are dissolved inpure chloroform (3 mL). This lipid solution is added to a 50 mLround-bottom flask, and the solvent is evaporated on a rotary evaporatorat a temperature of 60° C. The flask is then flushed with nitrogen gasto remove remaining solvent. Phosphate buffered saline (2 mL) and fiveglass beads are added to the flask, and the lipid film is hydrated byshaking at 60° C. for 1 h to form a suspension. The suspension istransferred to a small pressure tube and sonicated at 60° C. for fourcycles of 30 s pulses with a 30 s delay between each pulse. Thesuspension is then left undisturbed at room temperature for 2 h to allowfor complete hydration. The liposomes are washed by centrifugationfollowed by resuspension in fresh phosphate buffered saline.

Example 4 Polymeric Nanocarrier Containing Polymer-Rapamycin Conjugate(Prophetic)

Preparation of PLGA-Rapamycin Conjugate:

PLGA polymer with acid end group (7525 DLG1A, acid number 0.46 mmol/g,Lakeshore Biomaterials; 5 g, 2.3 mmol, 1.0 eq) is dissolved in 30 mL ofdichloromethane (DCM). N,N-Dicyclohexylcarbodimide (1.2 eq, 2.8 mmol,0.57 g) is added followed by rapamycin (1.0 eq, 2.3 mmol, 2.1 g) and4-dimethylaminopyridine (DMAP) (2.0 eq, 4.6 mmol, 0.56 g). The mixtureis stirred at rt for 2 days. The mixture is then filtered to removeinsoluble dicyclohexylurea. The filtrate is concentrated to ca. 10 mL involume and added to 100 mL of isopropyl alcohol (IPA) to precipitate outthe PLGA-rapamycin conjugate. The IPA layer is removed and the polymeris then washed with 50 mL of IPA and 50 mL of methyl t-butyl ether(MTBE). The polymer is then dried under vacuum at 35 C for 2 days togive PLGA-rapamycin as a white solid (ca. 6.5 g).

Preparation of Nanocarrier Containing PLGA-Rapamycin Conjugate andOvalbumin Peptide (323-339):

Nanocarrier containing PLGA-rapamycin is prepared according to theprocedure described in Example 1 as follows:

Solutions for nanocarrier formation are prepared as follows:

Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute hydrochloricacid aqueous solution. The solution is prepared by dissolving ovalbuminpeptide in 0.13 M hydrochloric acid solution at room temperature.Solution 2: PLGA-rapamycin @ 100 mg/mL in methylene chloride. Thesolution is prepared by dissolving PLGA-rapamycin in pure methylenechloride. Solution 3: PLA-PEG @ 100 mg/mL in methylene chloride. Thesolution is prepared by dissolving PLA-PEG in pure methylene chloride.Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphatebuffer.

A primary water-in-oil emulsion is prepared first. W1/O1 is prepared bycombining solution 1 (0.2 mL), solution 2 (0.75 mL), and solution 3(0.25 mL) in a small pressure tube and sonicating at 50% amplitude for40 seconds using a Branson Digital Sonifier 250. A secondary emulsion(W1/O1/W2) is then prepared by combining solution 4 (3.0 mL) with theprimary W1/O1 emulsion, vortexing for 10 s, and sonicating at 30%amplitude for 60 seconds using the Branson Digital Sonifier 250. TheW1/O1/W2 emulsion is added to a beaker containing 70 mM pH 8 phosphatebuffer solution (30 mL) and stirred at room temperature for 2 hours toallow the methylene chloride to evaporate and for the nanocarriers toform. A portion of the nanocarriers is washed by transferring thenanocarrier suspension to a centrifuge tube and centrifuging at 75,600×gand 4° C. for 35 min, removing the supernatant, and re-suspending thepellet in phosphate buffered saline. The washing procedure is repeated,and the pellet is re-suspended in phosphate buffered saline for a finalnanocarrier dispersion of about 10 mg/mL.

Example 5 Preparation of Gold Nanocarriers (AuNCs) Containing Rapamycin(Prophetic)

Preparation of HS-PEG-Rapamycin:

A solution of PEG acid disulfide (1.0 eq), rapamycin (2.0-2.5 eq), DCC(2.5 eq) and DMAP (3.0 eq) in dry DMF is stirred at rt overnight. Theinsoluble dicyclohexylurea is removed by filtration and the filtrate isadded to isopropyl alcohol (IPA) to precipitate out thePEG-disulfide-di-rapamycin ester and washed with IPA and dried. Thepolymer is then treated with tris(2-carboxyethyl)phosphine hydrochloridein DMF to reduce the PEG disulfide to thiol PEG rapamycin ester(HS-PEG-rapamycin). The resulting polymer is recovered by precipitationfrom IPA and dried as previously described and analyzed by H NMR andGPC.

Formation of Gold NCs (AuNCs):

An aq. solution of 500 mL of 1 mM HAuC14 is heated to reflux for 10 minwith vigorous stirring in a 1 L round-bottom flask equipped with acondenser. A solution of 50 mL of 40 mM of trisodium citrate is thenrapidly added to the stirring solution. The resulting deep wine redsolution is kept at reflux for 25-30 min and the heat is withdrawn andthe solution is cooled to room temperature. The solution is thenfiltered through a 0.8 μm membrane filter to give the AuNCs solution.The AuNCs are characterized using visible spectroscopy and transmissionelectron microscopy. The AuNCs are ca. 20 nm diameter capped by citratewith peak absorption at 520 nm.

AuNCs Conjugate with HS-PEG-Rapamycin:

A solution of 150 μl of HS-PEG-rapamycin (10 μM in 10 mM pH 9.0carbonate buffer) is added to 1 mL of 20 nm diameter citrate-capped goldnanocarriers (1.16 nM) to produce a molar ratio of thiol to gold of2500:1. The mixture is stirred at room temperature under argon for 1hour to allow complete exchange of thiol with citrate on the goldnanocarriers. The AuNCs with PEG-rapamycin on the surface is thenpurified by centrifuge at 12,000 g for 30 minutes. The supernatant isdecanted and the pellet containing AuNC-S-PEG-rapamycin is then pelletwashed with 1×PBS buffer. The purified Gold-PEG-rapamycin nanocarriersare then resuspend in suitable buffer for further analysis andbioassays.

Example 6 Mesoporous Silica-gold Core-shell Nanocarriers ContainingOvalbumin (Prophetic)

Mesoporous SiO2 nanoparticle cores are created through a sol-gelprocess. Hexadecyltrimethyl-ammonium bromide (CTAB) (0.5 g) is dissolvedin deionized water (500 mL), and then 2 M aqueous NaOH solution (3.5 mL)is added to the CTAB solution. The solution is stirred for 30 min, andthen Tetraethoxysilane (TEOS) (2.5 mL) is added to the solution. Theresulting gel is stirred for 3 h at a temperature of 80° C. The whiteprecipitate which forms is captured by filtration, followed by washingwith deionized water and drying at room temperature. The remainingsurfactant is then extracted from the particles by suspension in anethanolic solution of HCl overnight. The particles are washed withethanol, centrifuged, and redispersed under ultrasonication. This washprocedure is repeated two additional times.

The SiO2 nanoparticles are then functionalized with amino groups using(3-aminopropyl)-triethoxysilane (APTMS). To do this, the particles aresuspended in ethanol (30 mL), and APTMS (50 μL) is added to thesuspension. The suspension is allowed to stand at room temperature for 2h and then is boiled for 4 h, keeping the volume constant byperiodically adding ethanol. Remaining reactants are removed by fivecycles of washing by centrifugation and redispersing in pure ethanol.

In a separate reaction, 1-4 nm diameter gold seeds are created. Allwater used in this reaction is first deionized and then distilled fromglass. Water (45.5 mL) is added to a 100 mL round-bottom flask. Whilestirring, 0.2 M aqueous NaOH (1.5 mL) is added, followed by a 1% aqueoussolution of tetrakis(hydroxymethyl)phosphonium chloride (THPC) (1.0 mL).Two minutes after the addition of THPC solution, a 10 mg/mL aqueoussolution of chloroauric acid (2 mL), which has been aged at least 15min, is added. The gold seeds are purified through dialysis againstwater.

To form the core-shell nanocarriers, the amino-functionalized SiO2nanoparticles formed above are first mixed with the gold seeds for 2 hat room temperature. The gold-decorated SiO2 particles are collectedthrough centrifugation and mixed with an aqueous solution of chloroauricacid and potassium bicarbonate to form the gold shell. The particles arethen washed by centrifugation and redispersed in water. ThiolatedOvalbumin (made by treating Ovalbumin with 2-iminothiolanehydrochloride) is loaded by suspending the particles in a solution ofthiolated Ovalbumin (1 mg/L) for 72 h. The particles is then pelletwashed with 1×PBS (pH 7.4) to remove free protein. The resultingsilica-gold core-shell nanocarriers containing Ovalbumin are thenre-suspended in 1×PBS for further analysis and assays.

Example 7 Liposomes Containing Rapamycin and Ovalbumin (Prophetic)

The liposomes are formed by thin film hydration.1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (32 μmol),cholesterol (32 μmol), and rapamycin (6.4 μmol) are dissolved in purechloroform (3 mL). This lipid solution is added to a 10 mL glass tubeand the solvent is removed under nitrogen gas stream and desiccated for6 hr. under vacuum. Multilamellar vesicles are obtained by hydration ofthe film with 2.0 ml of 25 mM MOPS buffer pH 8.5, containing excessamount of Ovalbumin. The tube is vortexed until the lipid film is peeledof from the tube surface. To break the multilamellar vesicles intomonolamellar, ten cycles of freezing (liquid nitrogen) and thawing (30°C. water bath) are applied. The sample is then diluted to 1 ml in 25 mMMOPS buffer pH 8.5. Size of the resulting liposome is homogenized byextrusion by passing the sample 10 fold through a 200 nm porepolycarbonate filters. The resulting liposomes are then used for furtheranalysis and bioassays.

Example 8 Polymeric Nanocarriers Composed of Modified Polyamino Acidwith Surface Conjugated Ovalbumin (Prophetic)

Step-1. Preparation of Poly(γ-glutamic acid) (γ-PGA) modified withL-phenylalanine ethyl ester (L-PAE): 4.7 unit mmol of γ-PGA (Mn=300 kD)is dissolved in 0.3 N—NaHCO3 aqueous solution (50 mL). L-PAE (4.7 mmol)and EDC.HCl (4.7 mmol) are added to the solution and stirred for 30 minat 4 C. The solution is then maintained at room temperature withstirring for 24 h. Low-molecular-weight chemicals are removed bydialysis using dialysis membrane with MWCO 50 kD. The resultingγ-PGA-graft-L-PAE is obtained by freeze-drying.

Step-2. Preparation of nanoparticles from γ-PGA-graft-L-PAE polymer:Nanoparticles composed of γ-PGA-graft-L-PAE are prepared by aprecipitation and dialysis method. γ-PGA-graft-L-PAE (20 mg) wasdissolved in 2 ml of DMSO followed by addition of 2 mL of water to forma translucent solution. The solution is then dialyzed against distilledwater using cellulose membrane tubing (50,000 MWCO) to form thenanoparticles and to remove the organic solvents for 72 h at roomtemperature. The distilled water is exchanged at intervals of 12 h. Theresulting nanoparticle solution (10 mg/mL in water) is then used forantigen conjugation.

Step-3. Ovalbumin conjugation to γ-PGA nanoparticles: Surface carboxylicacid groups of the γ-PGA nanoparticles (10 mg/ml) are first activated byEDC and NHS (10 mg/mL each in phosphate buffer, pH 5.8) for 2 h atambient temperature. After pellet washing to remove excess EDC/NHS, theactivated nanoparticles are mixed with 1 mL of Ovalbumin (10 mg/ml) inphosphate-buffered saline (PBS, pH 7.4) and the mixture is incubated at4-8 C for 24 h. The resulting Ovalbumin conjugated γ-PGA nanoparticlesare washed twice with PBS and resuspended at 5 mg/mL in PBS for furtheranalysis and bioassays.

Example 9 Erythropoietin (EPO)-encapsulated γ-PGA Nanoparticles(Prophetic)

To prepare the EPO-encapsulated γ-PGA nanoparticles, 0.25-4 mg of EPO isdissolved in 1 mL of PBS (pH 7.4) and 1 mL of the γ-PGA-graft-L-PAE (10mg/mL in DMSO) is added to the EPO solution. The resulting solution iscentrifuged at 14,000×g for 15 min and repeatedly rinsed with PBS. Theresulting EPO-encapsulated γ-PGA nanoparticles are then resuspended inPBS (5 mg/mL) for further analysis and bioassay.

Example 10 Preparation of Gold Nanocarriers (AuNCs) Containing Ovalbumin(Prophetic)

Step-1. Formation of Gold NCs (AuNCs): An aq. solution of 500 mL of 1 mMHAuC14 is heated to reflux for 10 min with vigorous stirring in a 1 Lround-bottom flask equipped with a condenser. A solution of 50 mL of 40mM of trisodium citrate is then rapidly added to the stirring solution.The resulting deep wine red solution is kept at reflux for 25-30 min andthe heat is withdrawn and the solution is cooled to room temperature.The solution is then filtered through a 0.8 μm membrane filter to givethe AuNCs solution. The AuNCs are characterized using visiblespectroscopy and transmission electron microscopy. The AuNCs are ca. 20nm diameter capped by citrate with peak absorption at 520 nm.

Step-2. Conjugation of Ovalbumin to AuNCs: A solution of 150 μl ofthiolated Ovalbumin (10 μM in 10 mM pH 9.0 carbonate buffer) is added to1 mL of 20 nm diameter citrate-capped gold nanocarriers (1.16 nM) toproduce a molar ratio of thiol to gold of 2500:1. The mixture is stirredat room temperature under argon for 1 hour to allow complete exchange ofthiol with citrate on the gold nanocarriers. The AuNCs with Ovalbumin onthe surface is then purified by centrifuge at 12,000 g for 30 minutes.The supernatant is decanted and the pellet containing AuNC-Ovalbumin isthen pellet washed with 1×PBS buffer. The purified Gold-Ovalbuminnanocarriers are then resuspend in suitable buffer for further analysisand bioassays.

Example 11 Evaluating Tolerogenic Immune Response to Antigen In Vivo(Prophetic)

Balb/c mice are immunized with an antigen in incomplete Freund'sadjuvant, and the level of regulatory B cell proliferation is assessed.Subsequently, a composition of the invention is administered in adose-dependent manner. The same mice are then again exposed to theantigen, and the level of regulatory B cell proliferation is againassessed. Changes in regulatory B cell proliferation are then monitoredwith an increase upon subsequent challenge with the antigen indicating atolerogenic immune response.

Example 12 Evaluating In Vitro Induction of Regulatory B Cells(Prophetic)

A cell population comprising regulatory B cells or regulatory B cellprecursors is contacted in vitro with a composition provided herein.After a time sufficient for the composition to interact with theregulatory B cells or regulatory B cell precursors in the cellpopulation, an increase in the number of regulatory B cells is expected.A time sufficient for the increase in the number of regulatory B cellsin the population of cells is, in some embodiment, a period of about 1day, about 2 days, about 3 days, about 4 days, about 5 days, about 6days, about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. Insome embodiments, the number of regulatory B cells is increased afterthat time, for example, to at least about 5 times, at least about 10times, at least about 20 times, at least about 50 times, at least about100 times, at least about 1,000 times, at least about 10,000 times, atleast about 100,000 times, or at least about 1,000,000 times as comparedto the original total or relative number of regulatory B cells in thepopulation. In some embodiments, the number of regulatory B cells isincreased after that time, for example, to about 1%, about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%,about 75%, about 90%, or about 95% of the total cells in the populationor of the lymphocytes in the cell population. In some embodiments,regulatory B cells are absent from the cell population before thecontacting, but are present after the contacting.

In some embodiments, the total and/or relative number of regulatory Bcells in the population is determined before the population of cells iscontacted with a composition provided herein to establish a baselinenumber of regulatory B cells in the population. In some embodiments, thepopulation of cells is contacted once with a composition providedherein. In some embodiments, the population of cells is contactedrepeatedly with a composition provided herein.

The number and/or presence of regulatory B cells in the population ofcells is also determined after the contacting. In some embodiments, thenumber and/or presence of regulatory B cells is monitored over a periodof time, for example, by performing a plurality of subsequent regulatoryB cell detection assays. In some embodiments, the number and/or presenceof regulatory B cells in the population of cells is determined by takinga sample from the cell population that is representative of the cellpopulation, staining cells contained in that sample with antibodies orstaining agents that specifically detect regulatory B cell markers, anddetecting cells that express regulatory B cell markers in the sample,for example, by FACS or by immunohistochemistry. In some embodiments,the regulatory B cells are quantified. In some embodiments, the quantityof regulatory B cells determined after the contacting is compared to thequantity of regulatory B cells before the contacting, for example, tothe baseline number of regulatory B cells, wherein, if the number ofregulatory B cells in the population of cells is higher after thecontacting than the baseline number, then it is determined that atolerogenic response to the composition has occurred.

Example 13 Evaluating In Vivo Induction of Regulatory B Cells(Prophetic)

A cell population comprising regulatory B cells or regulatory B cellprecursors is contacted in vivo with a composition provided herein. Insome embodiments, the composition is administered to a subject harboringthe cell population comprising regulatory B cells or regulatory B cellprecursors. After a time sufficient for the composition to interact withthe regulatory B cells or regulatory B cell precursors in the subject,an increase in the number of regulatory B cells is expected. A timesufficient for the increase in the number of regulatory B cells in thepopulation is, in some embodiment, a period of about 1 day, about 2days, about 3 days, about 4 days, about 5 days, about 6 days, about 1week, about 2 weeks, about 3 weeks, or about 4 weeks. In someembodiments, the period of time sufficient to effect an increase in thenumber of regulatory B cells is longer than 4 weeks. In someembodiments, the number of regulatory B cells is increased after thattime to at least about 5 times, at least about 10 times, at least about20 times, at least about 50 times, at least about 100 times, at leastabout 1,000 times, at least about 10,000 times, at least about 100,000times, or at least about 1,000,000 times as compared to the originaltotal or relative number of regulatory B cells in the subject. In someembodiments, the number of regulatory B cells is increased after thattime, for example, to about 1%, about 5%, about 10%, about 15%, about20%, about 25%, about 30%, about 40%, about 50%, about 75%, about 90%,or about 95% of the total cells in the subject, or of a lymphocytepopulation in the subject. In some embodiments, regulatory B cells areabsent from the subject before the administration, but are present afterthe administration.

In some embodiments, the total and/or relative number of regulatory Bcells in the subject is determined before the subject is administered acomposition provided herein to establish a baseline number of regulatoryB cells in the subject. In some embodiments, the composition providedherein is administered to a subject once. In some embodiments, acomposition provided herein is administered to a subject multiple times,for example, until a desired increase or abundance of regulatory B cellsis observed in the subject.

The number and/or presence of regulatory B cells in the subject isdetermined after the administration. In some embodiments, the numberand/or presence of regulatory B cells is monitored over a period oftime, for example, by performing a plurality of subsequent regulatory Bcell detection assays. In some embodiments, the number and/or presenceof regulatory B cells in the subject is determined by taking a samplefrom the subject, for example, a peripheral blood sample, or a lymphsample, that is representative of a lymphocyte population in thesubject, staining cells contained in that sample with antibodies orstaining agents that specifically detect regulatory B cell markers, anddetecting cells that express regulatory B cell markers in the sample,for example, by FACS or by immunohistochemistry. In some embodiments,the regulatory B cells are quantified. In some embodiments, the quantityof regulatory B cells determined after administration is compared to thequantity of regulatory B cells before administration, for example, tothe baseline number of regulatory B cells, wherein, if the number ofregulatory B cells in the subject is higher after the administrationthan the baseline number, then it is determined that a tolerogenicresponse to the composition has occurred.

Example 14 In Vivo Induction of a Desired Immune Response AgainstTransplantable Bone Marrow Cells (Prophetic)

A population of at least 10⁶ synthetic nanocarriers comprisingimmunosuppressant and antigens obtained or derived from bone marrowcells are administered subcutaneously to a subject four weeks prior tothe subject receiving a bone marrow transplant. After the transplant isreceived by the subject, the generation of a desired immune response inthe subject is assessed once daily during the first week after receivingthe transplant, and then weekly for the next three weeks, and thenmonthly for the next 11 months. As part of the assessment, regulatory Bcell counts are taken and compared to regulatory B cell counts takenprior to administering the bone marrow transplant or the syntheticnanocarriers to the subject. During the first year, maintenance doses ofthe synthetic nanocarriers are administered bi-monthly to the subject.The subject is expected to exhibit higher or desired levels ofregulatory B cells specific to the bone marrow transplant.

Example 15 Assessing the Effects of Nanocarriers with Antigens andImmunosuppressants

Nanocarriers

Ovalbumin peptide 323-339, a 17 amino acid peptide known to be a T and Bcell epitope of Ovalbumin protein, was purchased from Bachem AmericasInc. (3132 Kashiwa Street, Torrance Calif. 90505; Part #4065609). PLGAwith a lactide:glycolide ratio of 3:1 and an inherent viscosity of 0.75dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive,Birmingham, Ala. 35211; Product Code 7525 DLG 7A). PLA-PEG blockco-polymer with a PEG block of approximately 5,000 Da and PLA block ofapproximately 20,000 Da was synthesized. Polyvinyl alcohol (85-89%hydrolyzed) was purchased from EMD Chemicals (Product Number1.41350.1001).

Solutions were prepared as follows: Solution 1: Ovalbumin peptide323-339 @ 20 mg/mL in dilute hydrochloric acid aqueous solution. Thesolution was prepared by dissolving ovalbumin peptide in 0.13 Mhydrochloric acid solution at room temperature. Solution 2: PLGA @ 100mg/mL in methylene chloride. The solution was prepared by dissolvingPLGA in pure methylene chloride. Solution 3: PLA-PEG @ 100 mg/mL inmethylene chloride. The solution was prepared by dissolving PLA-PEG inpure methylene chloride. Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100mM pH 8 phosphate buffer. A primary water-in-oil emulsion was preparedfirst. W1/O1 was prepared by combining solution 1 (0.2 mL), solution 2(0.75 mL), and solution 3 (0.25 mL) in a small pressure tube andsonicating at 50% amplitude for 40 seconds using a Branson DigitalSonifier 250. A secondary emulsion (W1/O1/W2) was then prepared bycombining solution 4 (3.0 mL) with the primary W1/O1 emulsion, vortexingfor 10 s, and sonicating at 30% amplitude for 60 seconds using theBranson Digital Sonifier 250.

The W1/O1/W2 emulsion was added to a beaker containing 70 mM pH 8phosphate buffer solution (30 mL) and stirred at room temperature for 2hours to allow the methylene chloride to evaporate and for thenanocarriers to form. A portion of the nanocarriers were washed bytransferring the nanocarrier suspension to a centrifuge tube andcentrifuging at 75,600×g and 4° C. for 35 min, removing the supernatant,and re-suspending the pellet in phosphate buffered saline. The washingprocedure was repeated, and the pellet was re-suspended in phosphatebuffered saline for a final nanocarrier dispersion of about 10 mg/mL.

Nanocarrier size was determined by dynamic light scattering. The amountof peptide in the nanocarrier was determined by HPLC analysis. The totaldry-nanocarrier mass per mL of suspension was determined by agravimetric method.

Effective Diameter Peptide Content Nanocarrier (nm) (% w/w) 1 234 2.1

Ovalbumin peptide 323-339, a 17 amino acid peptide known to be a T and Bcell epitope of Ovalbumin protein, was purchased from Bachem AmericasInc. (3132 Kashiwa Street, Torrance Calif. 90505; Part #4065609).Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham,Mass. 01702; Product Catalogue #R1017). PLGA with a lactide:glycolideratio of 3:1 and an inherent viscosity of 0.75 dL/g was purchased fromSurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211;Product Code 7525 DLG 7A). PLA-PEG block co-polymer with a PEG block ofapproximately 5,000 Da and PLA block of approximately 20,000 Da wassynthesized. Polyvinyl alcohol (85-89% hydrolyzed) was purchased fromEMD Chemicals (Product Number 1.41350.1001).

Solutions were prepared as follows: Solution 1: Ovalbumin peptide323-339 @ 20 mg/mL in dilute hydrochloric acid aqueous solution. Thesolution was prepared by dissolving ovalbumin peptide in 0.13 Mhydrochloric acid solution at room temperature. Solution 2: Rapamycin @50 mg/mL in methylene chloride. The solution was prepared by dissolvingrapamycin in pure methylene chloride. Solution 3: PLGA @ 100 mg/mL inmethylene chloride. The solution was prepared by dissolving PLGA in puremethylene chloride. Solution 4: PLA-PEG @ 100 mg/mL in methylenechloride. The solution was prepared by dissolving PLA-PEG in puremethylene chloride. Solution 5: Polyvinyl alcohol @ 50 mg/mL in 100 mMpH 8 phosphate buffer.

A primary water-in-oil emulsion was prepared first. W1/O1 was preparedby combining solution 1 (0.2 mL), solution 2 (0.2 mL), solution 3 (0.75mL), and solution 4 (0.25 mL) in a small pressure tube and sonicating at50% amplitude for 40 seconds using a Branson Digital Sonifier 250. Asecondary emulsion (W1/O1/W2) was then prepared by combining solution 5(3.0 mL) with the primary W1/O1 emulsion, vortexing for 10 s, andsonicating at 30% amplitude for 60 seconds using the Branson DigitalSonifier 250.

The W1/O1/W2 emulsion was added to a beaker containing 70 mM pH 8phosphate buffer solution (30 mL) and stirred at room temperature for 2hours to allow the methylene chloride to evaporate and for thenanocarriers to form. A portion of the nanocarriers were washed bytransferring the nanocarrier suspension to a centrifuge tube andcentrifuging at 21,000×g and 4° C. for 45 min, removing the supernatant,and re-suspending the pellet in phosphate buffered saline. The washingprocedure was repeated, and the pellet was re-suspended in phosphatebuffered saline for a final nanocarrier dispersion of about 10 mg/mL.

Nanocarrier size was determined by dynamic light scattering. The amountsof peptide and rapamycin in the nanocarrier were determined by HPLCanalysis. The total dry-nanocarrier mass per mL of suspension wasdetermined by a gravimetric method.

Effective Rapamycin Content Peptide Content Nanocarrier ID Diameter (nm)(% w/w) (% w/w) 2 227 9.0 2.5

Measurement of synthetic nanocarrier dimensions was obtained by dynamiclight scattering (DLS). A suspension of the synthetic nanocarriers wasdiluted with purified water to achieve a final synthetic nanocarriersuspension concentration of approximately 0.01 to 0.1 mg/mL. The dilutedsuspension was prepared directly inside a suitable cuvette for DLSanalysis. The cuvette was then placed in a Brookhaven Instruments Corp.ZetaPALS, allowed to equilibrate to 25° C., and then scanned forsufficient time to acquire a stable and reproducible distribution basedon appropriate inputs for viscosity of the medium and refractiveindicies of the sample. The effective diameter, or mean of thedistribution, was then reported.

Immunization

The nanocarriers were thawed and equilibrated. Initial dilutionsconstituted a 10× stock solution, and were further diluted to aconcentration of 100 μg/ml in OVA₃₂₃₋₃₃₉, or a 1× solution. This 1×solution was used for injections at 200 μl per i.v. injection. Animalswere immunized with OVA protein (OVA) and treated with OVA₃₂₃₋₃₃₉peptide. Immunization routes were as follows: 10 μg of OVA+4 mg Alumi.p. in 4000 per each Balb/C immunologically naïve female mouse.Experimental groups consisted of 5 animals each. Spleen cells wererestimulated with antigen using CFSE or CTO to determine the amount ofAg-specific proliferation.

Measuring Regulatory B Cells

The frequency of Ovalbumin reactive IL-10 secreting B cells wascalculated by way of flow cytometry. Splenocytes from experimentalanimals were stained with CFSE, a thiol-reactive Fluorescent Probesuitable for long-term cell labeling, and cultured in complete media at37 C, 5% CO2 with Ovalbumin protein for 3 days. On day 3 the cells wereassayed for their potential to secrete different cytokines byintracellular staining using standard methods and kits. Briefly, cellswere restimulated with phorbol myristate acetate (PMA) and lonomycin for2 hours and protein transport was blocked for another 4 hours.Unspecific binding of antibodies was blocked with anti-CD16/32 antibodyand then cells were stained with conjugated antibodies specificrecognizing specifically B cells (CD45R (B220) and CD19). After fixationwith paraformaldehyde cells were permeabilized to allow monoclonalantibodies into the cells and label intracellular epitopes (cytokines).Splenocytes that were B220+CD19+ were assessed for proliferation bycomparing the differential CFSE staining and the proportion ofIL-10-secreting cells determined.

Measurement of IgE Antibodies

IgE antibodies were measured using a Mouse OVA-IgE ELISA kit provided byMDBioproducts (Cat# M036005) consistent with the manufacturer'sinstructions.

Results

FIG. 2 demonstrate the effectiveness of the nanocarriers in thegeneration of regulatory B cells in lavage samples from animal subjectstreated with synthetic nanocarriers comprising OVA₃₂₃₋₃₃₉ andimmunosuppressant. The figure demonstrates that synthetic nanocarriersof the invention resulted in the production of IL-10 and TGF-β byantigen-specific CD24+ B cells. FIG. 3 shows a decrease in IgEproduction with synthetic nanocarriers of the invention.

Example 16 Isolation of Regulatory B Cell (Bregs) Cells from a Subjectafter Administration of Inventive Synthetic Nanocarriers (Prophetic)

Bregs are isolated from biological samples, for example, from peripheralblood, obtained from a subject after the subject is administered asynthetic nanocarrier composition as described herein. Typically, thebiological sample is obtained from the subject after a time periodsufficient for the administered itDCs to induce Bregs. Bregs areisolated from the biological sample, for example, from whole blood, bynegative and/or positive selection.

For example, the cellular fraction of whole blood is obtained bycentrifugation, and erythrocytes are lysed using erythrocyte lysisbuffer. After lysis, peripheral blood mononuclear cells are depleted forCD4+ cells and CD8+ T cells. Subsequently, Bregs are enriched for bypositive selection for the markers provided elsewhere herein orotherwise known in the art. In some embodiments, the isolation ofregulatory B cells is performed with a cocktail of biotinylatedantibodies and anti-biotin magnetic beads for the recognition of one ormore of these markers.

What is claimed is:
 1. A method for generating antigen-specificregulatory B cells that produce IL-10 in a mammalian subject,comprising: administering to the mammalian subject a composition thatcomprises: (i) a first population of synthetic nanocarriers coupled toimmunosuppressants, and (ii) a second population of syntheticnanocarriers coupled to antigens that comprise B cell epitopes and/orMHC Class II-restricted epitopes, wherein the composition is in anamount effective to generate antigen-specific regulatory B cells thatproduce IL-10 in the subject, and assessing the generation ofantigen-specific regulatory B cells that produce IL-10 in the subjectprior to and/or after the administration of the composition.
 2. A methodfor generating antigen-specific regulatory B cells that produce IL-10 ina mammalian subject, comprising: administering to the mammalian subjecta composition according to a protocol that was previously shown togenerate antigen-specific regulatory B cells that produce IL-10 in oneor more test subjects; wherein the composition comprises: (i) a firstpopulation of synthetic nanocarriers coupled to immunosuppressants, and(ii) a second population of synthetic nanocarriers coupled to antigensthat comprise B cell epitopes and/or MHC Class II-restricted epitopes.3. The method of claim 1 or 2, wherein the first population of syntheticnanocarriers and the second population of synthetic nanocarriers are thesame population.
 4. The method of claim 1 or 2, wherein the antigen isovalbumin, a therapeutic protein, an autoantigen, an allergen or anantigen associated with an inflammatory disease, an autoimmune disease,organ or tissue rejection or graft versus host disease.
 5. The method ofclaim 2, wherein the method further comprises assessing the generationof antigen-specific regulatory B cells that produce IL-10 in the subjectprior to and/or after the administration of the composition.
 6. Themethod of claim 1 or 2, wherein the subject has an inflammatory disease,an autoimmune disease, an allergy, organ or tissue rejection or graftversus host disease.
 7. The method of claim 1 or 2, wherein the subjecthas undergone or will undergo transplantation.
 8. The method of claim 1or 2, wherein the subject has an undesired immune response against atherapeutic protein that is being administered to the subject.
 9. Themethod of claim 1 or 2, wherein the immunosuppressants comprise astatin, an mTOR inhibitor, a TGF-β signaling agent, a corticosteroid, aninhibitor of mitochondrial function, a P38 inhibitor, an NF-κβinhibitor, an adenosine receptor agonist, a prostaglandin E2 agonist, aphosphodiesterase 4 inhibitor, an histone deacetylase (HDAC) inhibitoror a proteasome inhibitor.
 10. The method of claim 1 or 2, wherein theload of the immunosuppressants and/or epitopes on average across thefirst and/or second population of synthetic nanocarriers is between0.0001% and 50% (weight/weight).
 11. The method of claim 1 or 2, whereinthe synthetic nanocarriers of the first population and/or secondpopulation comprise lipid nanoparticles, polymeric nanoparticles,metallic nanoparticles, surfactant-based emulsions, dendrimers,buckyballs, nanowires, virus-like particles or peptide or proteinparticles.
 12. The method of claim 1 or 2, wherein the mean of aparticle size distribution obtained using dynamic light scattering ofthe synthetic nanocarriers of the first and/or second population is adiameter greater than 100 nm.
 13. The method of claim 1 or 2, whereinthe aspect ratio of the synthetic nanocarriers of the first populationand/or second population is greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3,1:5, 1:7 or 1:10.